Metabolically robust analogs of CYP-eicosanoids for the treatment of cardiac disease

ABSTRACT

The present invention relates to compounds according to general formula (I) which are metabolically robust analogues of bioactive lipid mediators derived from omega-3 polyunsaturated fatty acids (n-3 PUFAs). The present invention further relates to compositions containing one or more of these compounds and to the use of these compounds or compositions for the treatment or prevention of cardiovascular diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. national phase of International Application No. PCT/EP2016/067601, filed on Jul. 22, 2016, which claims priority to U.S. Provisional Application No. 62/195,645, filed Jul. 22, 2015, both of which are incorporated by reference herein in their entirety.

The present invention relates to compounds according to general formula (I) which are metabolically robust analogues of bioactive lipid mediators derived from omega-3 polyunsaturated fatty acids (n-3 PUFAs). The present invention further relates to compositions containing one or more of these compounds and to the use of these compounds or compositions for the treatment or prevention of cardiovascular diseases.

BACKGROUND OF THE INVENTION

Omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFAs) are essential components of the mammalian diet. Biologically most important n-3 PUFAs are eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). Dietary n-3 PUFAs have effects on diverse physiological processes impacting normal health and chronic disease, such as the regulation of plasma lipid levels, cardiovascular and immune function, inflammation, insulin action, and neuronal development and visual function.

Ingestion of n-3 PUFA will lead to their distribution to virtually every cell in the body with effects on membrane composition and function, eicosanoid synthesis, and signaling as well as the regulation of gene expression.

Epidemiological, clinical and experimental studies demonstrated that fish oil n-3 PUFAs (EPA and DHA) protect against cardiovascular disease. N-3 PUFAs reduce the mortality from coronary heart disease and the rate of sudden cardiac death.

Protection against ventricular arrhythmia is probably the main factor responsible for the prevention of sudden cardiac death by n-3 PUFAs after myocardial infarction and in heart failure patients. Significant antiarrhythmic effects of n-3 PUFAs were also observed in human studies on atrial fibrillation. The potential cardiac benefits from n-3 PUFAs extend further to the prevention and treatment of congestive heart failure and atherosclerosis as well as to the reduction of general risk factors such as high plasma levels of triglycerides and pro-inflammatory cytokines.

Additionally, epidemiological and experimental studies showed that n-3 PUFA consumption is associated with a reduced risk of macular degeneration and a lower incidence of colon, breast, prostate and other cancers. A major common mechanism in protecting against macular degeneration and cancer consists in the capacity of n-3 PUFAs to inhibit pathological angiogenesis. EPA and DHA inhibit vascular permeability, and inflammation. Angiogenesis is an essential step in tumor growth and metastasis that is promoted by n-6 PUFAs and n-6 PUFA-derived metabolites but inhibited by n-3 PUFAs and n-3 PUFA-derived metabolites.

Furthermore, one of the PUFAs most important biological roles is to supply precursors for the production of bioactive fatty acid metabolites that can modulate many functions. For instance, arachidonic acid (AA; 20:4, n-6) is metabolized by Cytochrome P450 (CYP) enzymes to several classes of oxygenated metabolites with potent biological activities. Major metabolites include 20-hydroxyeicosatetraenoic acid (20-HETE) and a series of regio- and stereoisomeric epoxyeicosatrienoic acids (EETs). CYP4A and CYP4F isoforms produce 20-HETE and CYP2C and CYP2J isoforms EETs.

It is known that EPA (20:5, n-3) and DHA (22:6, n-3) may serve as alternative substrates for AA-metabolizing CYP isoforms (Arnold C. et al., J Biol Chem. 2010 Oct. 22; 285(43):32720-33; Fischer R. et al., J Lipid Res. 2014 Mar. 16; 55(6):1150-1164.). CYP2C and CYP2J subfamily members that epoxidize AA to EETs, metabolize EPA to epoxyeicosatetraenoic acids (EEQs), and DHA to epoxydocosapentaenoic acids (EDPs). The ω-3 double bond distinguishing EPA and DHA from AA is the preferred site of attack by most of the epoxygenases resulting in the formation of 17,18-EEQ and 19,20-EDP as main metabolites. CYP4A and CYP4F isoforms, hydroxylating AA to 20-HETE, metabolize EPA to 20-hydroxyeicosapentaenoic acid (20-HEPE) and DHA to 22-hydroxydocosahexaenoic acid (22-HDHA). CYP1A1, CYP2E1 and other isoforms converting AA predominantly to 19-HETE show pronounced ω-3 epoxygenase activities with EPA and DHA. Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns. Cytochrome P450-dependent eicosapentaenoic acid metabolites are novel BK channel activators. A remarkable feature of CYP-dependent n-3 PUFA metabolism is the preferred epoxidation of the n-3 double bond, which distinguishes EPA and DHA from AA. The resulting metabolites—17,18-EEQ from EPA and 19,20-EDP from DHA—are unique in having no homolog within the series of AA products. In line with the substrate specificity of the CYP isoforms, dietary EPA/DHA supplementation causes a profound shift from AA- to EPA- and DHA-derived epoxy- and ω-hydroxy-metabolites in all major organs and tissues of the rat and presumably also in human.

EETs and 20-HETE play important roles in the regulation of various cardiovascular functions (Roman R J., Physiol Rev. 2002; 82:131-85). It has been shown that Ang II-induced hypertension is associated with a down-regulation of CYP-dependent AA metabolism (Kaergel et I., Hypertension. 2002; 40:273-9) in a double-transgenic rat (dTGR) model of Ang II-induced hypertension and end-organ damage (Luft et al., Hypertension. 1999; 33:212-8). The transgenic rats harbor the human renin and angiotensinogen genes, produce Ang II locally and develop significant hypertension, myocardial infarction and albuminuria. The animals die of myocardial and renal failure before the eighth week of age. The model shows severe features of Ang II-induced inflammation. Reactive oxygen species are generated, the transcription factors NE-κB and AP-1 are activated, and genes harboring binding sites for these transcription factors are activated.

Recently, it has been shown that eicosapentaenoic acid (EPA) supplementation significantly reduced the mortality of dTGR (Theuer et al., Kidney Int. 2005; 67:248-58). Additionally, it has been shown that dTGR develop ventricular arrhythmias based on Ang II-induced electrical remodeling (Fischer et sl. Am J Physiol Heart Circ Physiol. 2007; 293:H1242-1253). Treatment of the dTGR rats with a PPAR-alpha activator strongly induced CYP2C23-dependent EET production and protected against hypertension and end-organ damage (Muller et al., Am J Pathol. 2004; 164:521-32).

Long-term feeding of dTGR (from week 4 to 7 of age) with a mixture of pure EPA- and DHA-ethyl esters (Omacor from Solvay Arzneimittel, Hannover, Germany) improved the electrical remodeling of the heart in this model of angiotensin II-induced hypertension. In particular, EPA and DHA reduced the mortality, suppressed the inducibility of cardiac arrhythmias and protected against connexin 43-gap junctional remodeling (Fischer et al., Hypertension. 2008 February; 51(2):540-6). In general, CYP-dependent eicosanoids have to be considered as second messengers: EETs and 20-HETE are produced by CYP enzymes after extracellular signal induced release of AA from membrane phospholipids (by phospholipase A2) and exert their function in the context of signaling pathways modulating ion transport, cell proliferation and inflammation. Depending on the diet, n-3 PUFAs partially replace AA at the sn2-position of phospholipids and may thus become involved as alternative molecules in the subsequent signaling pathways.

The few studies on the biological activities of CYP-dependent eicosanoids in the heart indicate important roles for EETs and 20-HETE in the regulation of L-type Ca²⁺ and sarcolemmal and mitochondrial ATP-sensitive potassium (K_(ATP)) channels. In cardiac myocytes, L-type Ca²⁺ currents and cell shorting are reduced upon inhibition of EET generation and these effects can be reversed by adding 11,12-EET (Xiao et al., J Physiol. 1998; 508 (Pt 3):777-92). EETs were also shown to activate cardiac K_(ATP) channels. This effect is highly stereoselective: only the S,R but not the R,S-enantiomer of 11,12-EET was effective (Lu et al., Mol Pharmacol. 2002; 62:1076-83). Overexpression of the EET-generating human CYP2J2 resulted in an improved postischemic functional recovery of the transgenic mouse heart via activation of K_(ATP) channels (Seubert et al., Circ Res. 2004; 95:506-14). 20-HETE appears to play an opposite role by acting as an endogenous K_(ATP) channel blocker (Gross et al., J Mol Cell Cardiol. 2004; 37:1245-9; Nithipatikom et al., Circ Res. 2004; 95:e65-71).

The currently known biological activities of EPA- and DHA-derived CYP metabolites partially resemble those of their AA-derived counterparts, appear in part unique or may even produce opposite effects (Westphal et al., Prostaglandins Other Lipid Mediat. 2011; 96:99-108). The epoxymetabolites of all three PUFAs share vasodilatory properties, whereby the potencies of EEQs and EDPs may exceed those of EETs in some vascular beds (Lauterbach et al. Hypertension. 2002; 39:609-13). Anti-inflammatory effects were first revealed for 11,12- and 14,15-EET but are also exerted by EPA epoxides as exemplified by 17,18-EEQ (Morin et al., Am J Respir Cell Mol Biol. 2010; 43:564-575). 17,18-EEQ and 19,20-EDP inhibit the Ca²⁺— and isoproterenol-induced increased contractility of neonatal cardiomyocytes indicating that these metabolites may act as endogenous mediators of the antiarrhythmic effects of EPA and DHA described above (Arnold et al., J Biol Chem. 2010 Oct. 22; 285(43):32720-33). Chemically synthesized compounds were recently described that share the antiarrhythmic properties of 17,18-EEQ in neonatal cardiomyocytes and reduce ventricular tachyarrhythmia in a rat model of myocardial infarction (Falck et al., J Med Chem. 2011 Jun. 23; 54(12):4109-18; WO 2010/081683 A1, also published as US Pat. Pub. 2012/0122972). The formation of 17,18-EEQ and 19,20-EDP may additionally contribute to the anti-thrombotic effects of n-3 PUFAs (Jung et al., Clin Hemorheol Microcirc. 2012; 52(2-4):403-16). Moreover, there is evidence for an important role of CYP-dependent epoxymetabolites in mediating the opposite effects of n-6 and n-3 PUFAs in the processes of pathological angiogenesis described above, and thus, AA derived EETs promote tumor angiogenesis and metastasis (Panigrahy et al., J Clin Invest. 2012; 122:178-191). In contrast, 19,20-EDP and other regioisomeric DHA-epoxides inhibit these crucial events in cancerogenesis (Zhang et al., Proc Natl Acad Sci USA. 2013; 110:6530-6535).

Although n-3 PUFA-derived CYP metabolites, such as 17,18-EEQ and 19,20-EDP, play important roles in mediating the beneficial effects of n-3 PUFAs in the mammalian body, they are not used as therapeutics due to their limited bioavailability as well as chemical and metabolic instability. These epoxymetabolites of n-3 PUFAs are prone to autoxidation, rapid inactivation by the soluble epoxide hydrolase, and degradation by β-oxidation. Finally, new agents for the treatment or prevention of conditions and diseases associated with proliferation, pathological angiogenesis, hypertension, coagulation, immune function, heart failure and cardiac arrhythmias are of considerable interest as these conditions account for a significant number of death in patients and administration of many of the presently employed drugs is associated with complex drug interactions and many adverse side effects.

Therefore, the problem underlying the present invention is to provide new compounds, preferably new and improved analogues of n-3 PUFA metabolites. One problem underlying the present invention is the provision of improved compounds that are stable against deactivation by epoxide hydrolases, that are less prone to autooxidation, and which preferably have anti-atrial fibrillation, anti-ventricular arrhythmia, or anti-heart failure.

In a first aspect the above problem is solved by the provision of compounds of the general formula (I): P-E-I  (I) or a pharmaceutically acceptable salt thereof, wherein P is a group represented by the general formula (II): —(CH₂)_(n)—O—(CH₂)_(k)—X  (II) wherein n is 0 or an integer of from 3 to 8; and k is 0, 1 or 2; preferably with the proviso that when n is 0 k is 1, most preferably k is 1; X represents CH₂OH, CH₂OAc, CH(O) or a group selected from the group consisting of:

preferably X is

wherein R and R′ each independently represents a hydrogen atom; or a C₁-C₆alkyl group which may be substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R¹ represents a hydroxyl group, C₁-C₆alkoxy, —NHCN, —NH(C₁-C₆alkyl), —NH(C₃-C₆cycloalkyl), —NH(aryl), or —O(C₁-C₆alkyldiyl)O(C═O)R¹¹; R¹¹ is a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s); or a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R² represents —NHR³; —NR²⁰R²¹; —OR²²; —(OCH₂—CH₂)_(i)—R²³; —C₃-C₁₀-heterocyclyl optionally substituted with one, two or three substituents independently selected from the group consisting of hydroxyl group, C₁-C₆alkoxy, C₁-C₆alkyl, and oxo —(Xaa)_(o); a mono-, or disaccharide, or a derivative thereof, which is joined to —C(O) by an ester bond via the 1-O—, 3-O—, or 6-O-position of the saccharide; or is selected from the group consisting of:

wherein R³ represents (SO₂R³⁰); (OR³¹); —C₁-C₆alkanediyl(SO₂R³²); —C₁-C₆alkanediyl(CO₂H)), an aryl group, preferably phenyl, a heteroaryl group, preferably containing one ring and 5 or 6 ring atoms, a cycloalkyl group, preferably a C₃ to C₁₀-cycloalkyl, or a heterocycloalkyl group, preferably containing one or two ring systems, more preferably C₃ to C₁₀-heterocycloalkyl; wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and —C(═O)OR⁵¹; wherein the heteroaryl group, is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl and —C(═O)OR⁵¹; where the cycloalkyl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and —C(═O)OR⁵¹; and wherein the heterocycloalkyl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl and —C(═O)OR⁵¹; R³⁰ is a C₁-C₆alkyl, or an aryl group, wherein the C₁-C₆alkyl group is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, one, two or three fluorine or chlorine atoms, or a hydroxyl group; and wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), and —N(C₁-C₆)dialkyl; R³¹ is a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); or a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R³² is a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); or a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R²⁰ and R²¹ each independently represents a hydrogen atom; a C₁-C₆alkyl group which may be substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); a C₃-C₆cycloalkyl group which may be substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); or —C₁-C₆alkyldiyl(CO₂H) or together form a C₃-C₁₀-heterocycloalkyl, preferably a C₅-C₆-heterocycloalkyl, wherein the C₃-C₁₀-heterocycloalkyl may be substituted with one or more C₁-C₆alkyl group(s), C₁-C₆alkoxy group(s), fluorine or chlorine atom(s) or hydroxyl group(s); R²² is a hydrogen atom, a C₁-C₆alkyl group; or a C₃-C₆cycloalkyl group; wherein the C₁-C₆alkyl group or the C₃-C₆cycloalkyl group is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxyl, or C₁-C₆alkoxy, an aralkyl group, a heteroalkyl group or a heteroalkylcycloalkyl group; R²³ is —OH, —O(C₁-C₃)alkyl, or —N(C₁-C₃)dialkyl; i is an integer of from 1 to 10, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2 to 4; R²⁴, R²⁵, and R²⁶ each independently represents a hydrogen atom; —C(═O)C₁₁-C₂₁alkyl; or —C(═O)C₁₁-C₂₁alkenyl; R²⁷ represents —OH; —O(CH₂)₂NH₂, —OCH₂—[CH(NH₂)(CO₂H)], —O(CH₂)₂N(CH₃)₃; or

Xaa represents Gly, a conventional D,L-, D- or L-amino acid, a non-conventional D,L-, D- or L-amino acid, or a 2- to 10-mer peptide; and is joined to —C(═O) by an amide bond; o is an integer of from 1 to 10, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R⁴ is selected from the group consisting of:

h is 0, 1, or 2;

R⁵ represents a hydrogen atom; a fluorine or chlorine atom; —CF₃; —C(═O)OR⁵¹;

—NHC(═O)R⁵²; —C(═O)NR⁵³R⁵⁴; or —S(O₂)OH;

R⁵¹ represents a hydrogen atom; a C₁-C₆alkyl group; or a C₃-C₆cycloalkyl group; wherein the C₁-C₆alkyl group or the C₃-C₆cycloalkyl group is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxyl, or C₁-C₆alkoxy; R⁵², R⁵³ and R⁵⁴ each independently represents a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s); a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s); or an aryl group which is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent; R⁶ and R⁷ each independently represents a hydroxyl group; an —O(C₁-C₆)alkyl group, an —O(C₂-C₆)alkenyl group, a, —O(C₁-C₆)alkyldiylO(C═O)(C₁-C₆)alkyl group, or a —O(C₁-C₆)alkyldiylO(C═O)(C₂-C₆)alkenyl group; wherein the C₁-C₆alkyl group and the C₂-C₆alkenyl group may be substituted with NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆ alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, or one, two or three fluorine or chlorine atom(s); or R⁶ represents a hydroxyl group and R⁷ represents a group:

R⁹ represents C₁-C₆alkyl, or aryl; wherein the C₁-C₆alkyl is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxy, C₁-C₆alkoxy, aryl, aryloxy, —C(═O)-aryl, —C(═O)C₁-C₆alkoxy; and wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent; g is 1 or 2; X¹ represents an oxygen atom; sulfur atom; or NH; X² represents an oxygen atom; sulfur atom; NH; or N(CH₃); X³ represents an oxygen atom; sulfur atom; nitrogen atom; carbon atom; or C—OH; and the dashed line represents a carbon-carbon bond or a carbon-carbon double bond; E is a group represented by the general formula (III) or (IV):

wherein R¹² and R¹³ are preferably in cis configuration, and wherein ring A in formula (III) represents a 5-membered or 6-membered carbocyclic or heterocyclic ring containing at least one double bond, including an aromatic carbocyclic or heterocyclic ring, which can be substituted with one to three or one to four substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), and —N(C₁-C₆)dialkyl; and L and T each independently represents a ring atom, wherein L and T are adjacent to another; R¹² and R¹³ each independently represents a hydrogen atom, a fluorine atom, hydroxyl, —NH₂, C₁-C₆alkyl, C₁-C₆alkoxy, —C(═O)-aryl, —C(═O)C₁-C₆alkyl, or —SO₂(C₁-C₆alkyl); or —SO₂aryl; wherein any of the foregoing C₁-C₆alkyl, C₁-C₆alkoxy, or aryl are optionally substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; or R¹² and R¹³ are taken together to form a 5-membered or 6-membered ring, which ring is optionally substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl;

I is —(CH₂)_(m)—Y, wherein

m is an integer of from 3 to 6, provided that m is an integer of from 3 to 5 when E is a group according to general formula (III);

Y represents —U—V—W—(CH₂)_(p)—(CH₃)_(q), wherein p is an integer from 0 to 6; q is 0 or 1; U is absent or selected from the group consisting of CH, CH₂ and NR⁴⁰, with the proviso that U is only CH if it forms an epoxy group together with V and W; V is selected from the group consisting of —C(O)—, —C(O)—C(O)—, —O—, and —S—; W is selected from the group consisting of CH, CH₂ and NR⁴⁰ with the proviso that W is only CH if it forms an epoxy group together with U and V; or Y represents a group selected from the group consisting of:

wherein R⁴⁰, R⁴¹, R⁴², R⁴³, R⁴⁴, R⁴⁶, R⁴⁸ and R⁴⁹ each independently represents a hydrogen atom, —C₁-C₆alkyl, —C₃-C₆cycloalkyl, —C₁-C₆alkoxy, —C(═O)aryl, or —C(═O)C₁-C₆alkyl, wherein any of the foregoing C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆alkoxy, or aryl are optionally substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxy; or R⁴⁰ and R⁴¹, or R⁴³ and R⁴⁴, are taken together to form a 5-membered or 6-membered ring, which ring may be substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; R⁴², R⁴⁵, R⁴⁷ and R⁵⁰ each independently represents a —C₁-C₃alkyl, wherein the C₁-C₃alkyl may be substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₃)alkyl, —N(C₁-C₃)dialkyl, C₁-C₃alkylcarbonyloxy-, C₁-C₃alkoxycarbonyloxy-, C₁-C₃alkylcarbonylthio-, C₁-C₃alkylaminocarbonyl-, di(C₁-C₃)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; or R⁴⁰ and R⁴¹; R⁴³ and R⁴⁴, R⁴⁹ and R⁵⁰ are taken together to form a 5-membered or 6-membered ring, which ring may be substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; f is an integer of from 0 to 2; with the proviso that when X does not comprise a —C(═O)O-motif with the carbonyl carbon in alpha or beta position to the oxygen atom of general formula (II), Y is an oxamide, a carbamate or a carbamide, preferably Y is an oxamide as defined above. In a preferred embodiment, the compounds of present invention are compounds as described above with the further proviso that when n is 3, 5, 6, 7 or 8, k is 1 and E is a group according to general formula (III) or general formula (IV), wherein each of R¹² and R¹³ is a hydrogen atom; P represents a group: —(CH₂)₃—O—(CH₂)—X⁸¹; —(CH₂)₅—O—(CH₂)—X⁸¹; wherein X⁸¹ represents a group selected from the group consisting of:

R^(1′) is defined as R¹ above;

R^(2′) represents —NHR^(3′); —OR^(22′); —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O—, 3-O—, or 6-O-position of the saccharide;

or wherein R² is selected from the group consisting of:

wherein R³ represents (SO₂R³⁰); (OR³¹); —C₁-C₆alkanediyl(SO₂R³²); or —C₂-C₆alkanediyl(CO₂H); R^(22′) is a hydrogen or a C₃-C₆cycloalkyl group, which is optionally substituted with —NH2, —NH(C₁-C₆)alkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxy, or C₁-C₆alkoxy; R²³ and i are as defined above; R²⁴, R²⁵, R²⁶, and R²⁷ are as defined above; R^(4′) is defined as R⁴ above; and h is defined as above; R^(6′) and R^(7′) are defined as R⁶ and R⁷ above; R^(8″) and R^(8″) are defined as R⁸ and R^(8′) above; R^(9′) is defined as R⁹ above; R^(9″) represents aryl which is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent.

In a more preferred embodiment the compound of the present invention is one, wherein X is

wherein R² is —OR²²; —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O—, 3-O—, or 6-O-position of the saccharide; or wherein R² is selected from the group consisting of:

wherein R²³ and i are as defined above, preferable i is 3; and wherein R²², and R²³ to R²⁷ are as defined in claim 1, preferably R²² is a hydrogen atom or a C₁-C₆alkyl group, more preferably a hydrogen atom.

In one embodiment R₃ is not an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocycloalkyl group.

In one embodiment R²⁰ and R²¹ do not form together a C₃-C₁₀-heterocycloalkyl.

In one embodiment X is not

In one embodiment R₂ is not —C₃-C₁₀-heterocyclyl.

In a further preferred embodiment, the compound of the present invention is one, wherein X is

more preferably,

In this embodiment it is further preferred that Y is one of the oxamides defined above.

In a further more preferred embodiment, the compound of the present invention is one, wherein X is —C(═O)OH or a suitable salt of the carboxylic acid, preferably a free carboxylic acid.

In a further more preferred embodiment, the compound of the present invention is one, wherein X is

In this embodiment it is particularly preferred that g is 2. Another preferred embodiment of this preferred embodiment is one in which R⁹ represents C₁-C₆alkyl, i.e. methyl, ethyl, propyl, butyl, pentyl or hexyl, preferably methyl, or aryl, preferably phenyl; wherein the C₁-C₆alkyl is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxy, C₁-C₆alkoxy, aryl, aryloxy, —C(═O)-aryl, —C(═O)C₁-C₆alkoxy; and wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent. In this embodiment k is preferably 1 or 2; more preferably k is 1. In this case it is preferred that n is 0. In this embodiment it is further preferred that Y is one of the oxamides defined above.

In a further more preferred embodiment, the compound of the present invention is one, wherein X is

In this embodiment the hydroxy group may be in para, meta or ortho position, preferably in para position. In this embodiment is also preferred that R⁵ is hydrogen. In this embodiment it is further preferred that Y is one of the oxamides defined above.

In another more preferred embodiment, the compound of the present invention is one, wherein Y is one of the oxamides as defined above.

It is further preferred that the compound of the present invention is one, wherein X is

wherein R² is —OR²²; —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O—, 3-O—, or 6-O-position of the saccharide; or wherein R² is selected from the group consisting of:

wherein and R²², R²³ to R²⁷ and i are as defined above, preferably R²² is a hydrogen atom or a C₁-C₆alkyl group, more preferably a hydrogen atom, preferably i is 2 to 4, more preferably 3, and wherein Y is preferably one of the oxamides defined above.

It is further preferred that the compound of the present invention is one, wherein X is

and wherein R² is —C₃-C₁₀-heterocyclyl optionally substituted with one, two or three substituents independently selected from the group consisting of hydroxyl group, C₁-C₆alkoxy, C₁-C₆alkyl, and oxo.

In a more preferred embodiment, the compound of the present invention is one, wherein X is C(═O)OH, preferably the free carboxylic acid, and Y is preferably one of the oxamides defined above.

In another more preferred embodiment, the compound of the present invention is one with the following formula (V):

wherein R⁵⁵ represents —OH; —OR²²; —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O—, 3-O—, or 6-O-position of the saccharide; R²², R²³ and i are as defined above, preferably R²² is a hydrogen atom or a C₁-C₆alkyl group, more preferably a hydrogen atom and i is preferably 2 to 4, more preferably 3; Y represents a group selected from the group consisting of:

wherein R⁴⁰ to R⁵⁰ are defined above, preferably R⁴⁰ is a hydrogen atom or a C₁-C₆alkyl group, more preferably a hydrogen atom R⁵⁷ and R⁵⁸ are hydrogen; or form together a five- or six-membered ring, preferably an aromatic ring, optionally substituted with one to three or one to four substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent; s is 0, 1 or 2, with the proviso that s is 0 if R⁵⁷ and R⁵⁸ form together a five- or six-membered ring; the double bond in formula (V) represents a double carbon-carbon bond in cis-configuration, if R⁵⁷ and R⁵⁸ are hydrogen, or this double bond is part of a five- or six-membered ring formed together by R⁵⁷ and R⁵⁸. In a further most preferred embodiment the compounds of formula (V) are those wherein R⁵⁵ represents —OH or —(OCH₂—CH₂)_(i)—R²³; i is 2 to 4, preferably i is 3; R²³ is preferably OH; Y is an oxamide, a carbamide or a carbamate, preferably a C₁-C₆alkyl substituted oxamide, carbamide or carbamate; R⁵⁷ and R⁵⁸ are both H, or together form a substituted or non-substituted five- or six-membered aromatic ring, preferably form a substituted or non-substituted benzyl ring; and s is 1 or s is 0 if R⁵⁷ and R⁵⁸ together form a substituted or non-substituted five- or six-membered aromatic ring.

The most preferred specific compounds of the present invention are those selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

Among the above, the compound with the following formula (VI)

or a pharmaceutically acceptable salt thereof is most preferred.

The compounds of the present invention have the advantage as demonstrated below in the experimental section that they are effective for treating cardiac diseases. They are at the same time metabolically robust for pharmaceutical formulation and administration to subjects in need thereof.

The compounds described herein are generally described using standard nomenclature. For compounds having asymmetric centers, it is understood that, unless otherwise specified, all of the optical isomers and mixtures thereof are encompassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers. In addition, compounds with carbon-carbon double bonds may occur in Z- and E-forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. Recited compounds are further intended to encompass compounds in which one or more atoms are replaced with an isotope, i.e., an atom having the same atomic number but a different mass number. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include ¹¹C, ¹³C, and ¹⁴C.

Compounds according to the formulas provided herein, which have one or more stereogenic center(s), have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors, biosynthesis, e.g. using modified CYP102 (CYP BM-3) or by resolution of the racemates, e.g. enzymatic resolution or resolution by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.

Certain compounds are described herein using a general formula that includes variables such as, e.g. P, E, I, R¹-R⁵⁰, X-X⁸¹, and Y. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R*, the group may be unsubstituted or substituted with up to two R* groups, and R* at each occurrence is selected independently from the definition of R*. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested for biological activity.

A “pharmaceutically acceptable salt” of a compound disclosed herein is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.

Suitable pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC—(CH₂)_(n)—COOH where n is any integer from 0 to 6, i.e. 0, 1, 2, 3, 4, 5 or 6, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein. In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.

It will be apparent that each compound of formula (I) may, but need not, be present as a hydrate, solvate or non-covalent complex. In addition, the various crystal forms and polymorphs are within the scope of the present invention as are prodrugs of the compounds of formula (I) provided herein.

A “prodrug” is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound of formula (I) provided herein. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.

A “substituent,” as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a “ring substituent” may be a moiety such as a halogen, alkyl group, haloalkyl group or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term “substituted,” as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity. When a substituent is oxo, i.e., ═O, then 2 hydrogens on the atom are replaced. An oxo group that is a substituent of an aromatic carbon atom results in a conversion of —CH— to —C(═O)— and a loss of aromaticity. For example a pyridyl group substituted by oxo is a pyridone.

The expression “optionally substituted” refers to a group in which one, two, three or more hydrogen atoms may have been replaced independently of each other by the respective substituents.

As used herein, the term “amino acid” refers to any organic acid containing one or more amino substituents, e.g. α-, β- or γ-amino, derivatives of aliphatic carboxylic acids. In the polypeptide notation used herein, e.g. Xaa₅, i.e. Xaa₁Xaa₂Xaa₃Xaa₄Xaa₅, wherein Xaa₁ to Xaa₅ are each and independently selected from amino acids as defined, the left hand direction is the amino terminal direction and the right hand direction is the carboxy terminal direction, in accordance with standard usage and convention.

The term “conventional amino acid” refers to the twenty naturally occurring amino acids, and encompasses all stereomeric isoforms, i.e. D, L-, D- and L-amino acids thereof. These conventional amino acids can herein also be referred to by their conventional three-letter or one-letter abbreviations and their abbreviations follow conventional usage (see, for example, Immunology—A Synthesis, 2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)).

The term “non-conventional amino acid” refers to unnatural amino acids or chemical amino acid analogues, e.g. α,α-disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), and ortho-, meta- or para-aminobenzoic acid. Non-conventional amino acids also include compounds which have an amine and carboxyl functional group separated in a 1,3 or larger substitution pattern, such as β-alanine, γ-amino butyric acid, Freidinger lactam, the bicyclic dipeptide (BTD), amino-methyl benzoic acid and others well known in the art. Statine-like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art may also be used. The use of analogues or non-conventional amino acids may improve the stability and biological half-life of the added peptide since they are more resistant to breakdown under physiological conditions. The person skilled in the art will be aware of similar types of substitution which may be made. A non limiting list of non-conventional amino acids which may be used as suitable building blocks for a peptide and their standard abbreviations (in brackets) is as follows: α-aminobutyric acid (Abu), L-N-methylalanine (Nmala), α-amino-α-methylbutyrate (Mgabu), L-N-methylarginine (Nmarg), aminocyclopropane (Cpro), L-N-methylasparagine (Nmasn), carboxylate L-N-methylaspartic acid (Nmasp), aniinoisobutyric acid (Aib), L-N-methylcysteine (Nmcys), aminonorbornyl (Norb), L-N-methylglutamine (Nmgln), carboxylate L-N-methylglutamic acid (Nmglu), cyclohexylalanine (Chexa), L-N-methylhistidine (Nmhis), cyclopentylalanine (Cpen), L-N-methylisolleucine (Nmile), L-N-methylleucine (Nmleu), L-N-methyllysine (Nmlys), L-N-methylmethionine (Nmmet), L-N-methylnorleucine (Nmnle), L-N-methylnorvaline (Nmnva), L-N-methylornithine (Nmorn), L-N-methylphenylalanine (Nmphe), L-N-methylproline (Nmpro), L-N-methylserine (Nmser), L-N-methylthreonine (Nmthr), L-N-methyltryptophan (Nmtrp), D-ornithine (Dorn), L-N-methyltyrosine (Nmtyr), L-N-methylvaline (Nmval), L-N-methylethylglycine (Nmetg), L-N-methyl-t-butylglycine (Nmtbug), L-norleucine (Nle), L-norvaline (Nva), α-methylaminoisobutyrate (Maib), α-methyl-γ-aminobutyrate (Mgabu), D-α-methylalanine (Dmala), α-methylcyclohexylalanine (Mchexa), D-α-methylarginine (Dmarg), α-methylcylcopentylalanine (Mcpen), D-α-methylasparagine (Dmasn), α-methyl-α-napthylalanine (Manap), D-α-methylaspartate (Dmasp), α-methylpenicillamine (Mpen), D-α-methylcysteine (Dmcys), N-(4-aminobutyl)glycine (Nglu), D-α-methylglutamine (Dmgln), N-(2-aminoethyl)glycine (Naeg), D-α-methylhistidine (Dmhis), N-(3-aminopropyl)glycine (Norn), D-α-methylisoleucine (Dmile), N-amino-α-methylbutyrate (Nmaabu), D-α-methylleucine (Dmleu), α-napthylalanine (Anap), D-α-methyllysine (Dmlys), N-benzylglycine (Nphe), D-α-methylmethionine (Dmmet), N-(2-carbamylethyl)glycine (Ngln), D-α-methylornithine (Dmorn), N-(carbamylmethyl)glycine (Nasn), D-α-methylphenylalanine (Dmphe), N-(2-carboxyethyl)glycine (Nglu), D-α-methylproline (Dmpro), N-(carboxymethyl)glycine (Nasp), D-α-methylserine (Dmser), N-cyclobutylglycine (Ncbut), D-α-methylthreonine (Dmthr), N-cycloheptylglycine (Nchep), D-α-methyltryptophan (Dmtrp), N-cyclohexylglycine (Nchex), D-α-methyltyrosine (Dmty), N-cyclodecylglycine (Ncdec), D-α-methylvaline (Dmval), N-cylcododecylglycine (Ncdod), D-N-methylalanine (Dnmala), N-cyclooctylglycine (Ncoct), D-N-methylarginine (Dnmarg), N-cyclopropylglycine (Ncpro), D-N-methylasparagine (Dnmasn), N-cycloundecylglycine (Ncund), D-N-methylaspartate (Dnmasp), N-(2,2-diphenylethyl)glycine (Nbhm), D-N-methylcysteine (Dnmcys), N-(3,3-diphenylpropyl)glycine (Nbhe), D-N-methylglutamine (Dnmgln), N-(3-guanidinopropyl)glycine (Narg), D-N-methylglutamate (Dnmglu), N-(1-hydroxyethyl)glycine (Ntbx), D-N-methylhistidine (Dnmhis), N-(hydroxyethyl))glycine (Nser), D-N-methylisoleucine (Dnmile), N-(imidazolylethyl))glycine (Nhis), D-N-methylleucine (Dnmleu), N-(3-indolylyethyl)glycine (Nhtrp), D-N-methyllysine (Dnnilys), N-methyl-γ-aminobutyrate (Nmgabu), N-methylcyclohexylalanine (Nmchexa), D-N-methylmethionine (Dnmmet), D-N-methylornithine (Dnmorn), N-methylcyclopentylalanine (Nmcpen), N-methylglycine (Nala), D-N-methylphenylalanine (Dnmphe), N-methylaminoisobutyrate (Nmaib), D-N-methylproline (Dnmpro), N-(1-methylpropyl)glycine (Nile), D-N-methylserine (Dnmser), N-(2-methylpropyl)glycine (Nleu), D-N-methylthreonine (Dnmthr), D-N-methyltryptophan (Dnmtrp), N-(1-methylethyl)glycine (Nval), D-N-methyltyrosine (Dnmtyr), N-methyla-napthylalanine (Nmanap), D-N-methylvaline (Dnmval), N-methylpenicillamine (Nmpen), γ-aminobutyric acid (Gabu), N-(p-hydroxyphenyl)glycine (Nhtyr), L−/− butylglycine (Tbug), N-(thiomethyl)glycine (Ncys), L-ethylglycine (Etg), penicillamine (Pen), L-homophenylalanine (Hphe), L-α-methylalanine (Mala), L-α-methylarginine (Marg), L-α-methylasparagine (Masn), L-α-methylaspartate (Masp), L-α-methyl-t-butylglycine (Mtbug), L-α-methylcysteine (Mcys), L-methylethylglycine (Metg), L-α-methylglutamine (Mgln), L-α-methylglutamate (Mglu), L-α-methylhistidine (Mhis), L-α-methylhomophenylalanine (Mhphe), L-α-methylisoleucine (Mile), N-(2-methylthioethyl)glycine (Nmet), L-α-methylleucine (Mleu), L-α-methyllysine (Mlys), L-α-methylmethionine (Mmet), L-α-methylnorleucine (Mnle), L-α-methylnorvaline (Mnva), L-α-methylornithine (Morn), L-α-methylphenylalanine (Mphe), L-α-methylproline (Mpro), L-α-methylserine (Mser), L-α-methylthreonine (Mthr), L-α-methyltryptophan (Mtrp), L-α-methyltyrosine (Mtyr), L-α-methylvaline (Mval), L-N-methylhomophenylalanine (Nmhphe), N—(N-(2,2-diphenylethyl)carbamylmethyl)glycine (Nnbhm), N—(N-(3,3-diphenylpropyl)-carbamylmethyl)glycine (Nnbhe), 1-carboxy-1-(2,2-diphenyl-ethylamino)cyclopropane (Nmbc), L-O-methyl serine (Omser), L-O-methyl homoserine (Omhser).

The expression alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, e.g. a n-octyl group, especially from 1 to 6, i.e. 1, 2, 3, 4, 5, or 6, carbon atoms, for example a methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, or 2,2-dimethylbutyl.

The expression alkenyl refers to an at least partially unsaturated, straight-chain or branched, hydrocarbon group that contains from 2 to 21 carbon atoms, preferably from 2 to 6 carbon atoms, i.e. 2, 3, 4, 5 or 6 carbon atoms, for example an ethenyl (vinyl), propenyl (allyl), iso-propenyl, butenyl, isoprenyl or hex-2-enyl group, or from 11 to 21 carbon atoms, i.e. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 carbon atoms, for example a hydrocarbon group comprising a methylene chain interrupted by one double bond as, for example, found in monounsaturated fatty acids or a hydrocarbon group comprising methylene-interrupted polyenes, e.g. hydrocarbon groups comprising two or more of the following structural unit —[CH═CH—CH₂]—, as, for example, found in polyunsaturated fatty acids. Alkenyl groups have one or more, preferably 1, 2, 3, 4, 5, or 6 double bond(s).

The expression alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 10 carbon atoms, especially from 2 to 6, i.e. 2, 3, 4, 5 or 6, carbon atoms, for example an ethinyl, propinyl, butinyl, acetylenyl, or propargyl group. Preferably, alkynyl groups have one or two (especially preferably one) triple bond(s).

Furthermore, the terms alkyl, alkenyl and alkynyl refer to groups in which one or more hydrogen atom(s) have been replaced, e.g. by a halogen atom, preferably F or Cl, such as, for example, a 2,2,2-trichloroethyl or a trifluoromethyl group.

The expression heteroalkyl refers to an alkyl, alkenyl or alkynyl group in which one or more, preferably 1, 2 or 3, carbon atoms, have been replaced independently of each other by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom, preferably by an oxygen, sulfur or nitrogen atom. The expression heteroalkyl can also refer to a carboxylic acid or to a group derived from a carboxylic acid, such as, for example, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide or alkoxycarbonyloxy.

Preferably, a heteroalkyl group contains from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from oxygen, nitrogen and sulphur (especially oxygen and nitrogen). Especially preferably, a heteroalkyl group contains from 1 to 6, i.e. 1, 2, 3, 4, 5, or 6, carbon atoms and 1, 2 or 3, especially 1 or 2, hetero atoms selected from oxygen, nitrogen and sulphur, especially oxygen and nitrogen.

Examples of heteroalkyl groups are groups of formulae: R^(a)—O—Y^(a)—, R^(a)—S—Y^(a)—, R^(a)—N(R^(b))—Y^(a)—, R^(a)—CO—Y^(a)—, R^(a)—O—CO—Y^(a)—, R^(a)—CO—O—Y^(a)—, R^(a)—CO—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CO—Y^(a)—, R^(a)—O—CO—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CO—O—Y^(a)—, R^(a)—N(R^(b))—CO—N(R^(c))—Y^(a)—, R^(a)—O—CO—O—Y^(a)—, R^(a)—N(R^(b))—C(═NR^(d))—N(R^(c))—Y^(a)—, R^(a)—CS—Y^(a)—, R^(a)—O—CS—Y^(a)—, R^(a)—CS—O—Y^(a)—, R^(a)—CS—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CS—Y^(a)—, R^(a)—O—CS—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CS—O—Y^(a)—, R^(a)—N(R^(b))—CS—N(R^(c))—Y^(a)—, R^(a)—O—CS—O—Y^(a)—, R^(a)—S—CO—Y^(a)—, R^(a)—CO—S—Y^(a)—, R^(a)—S—CO—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CO—S—Y^(a)—, R^(a)—S—CO—O—Y^(a)—, R^(a)—O—CO—S—Y^(a)—, R^(a)—S—CO—S—Y^(a)—, R^(a)—S—CS—Y^(a)—, R^(a)—CS—S—Y^(a)—, R^(a)—S—CS—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CS—S—Y^(a)—, R^(a)—S—CS—O—Y^(a)—, R^(a)—O—CS—S—Y^(a)—, wherein R^(a) being a hydrogen atom, a C₁-C₆ alkyl, a C₂-C₆ alkenyl or a C₂-C₆ alkynyl group; R^(b) being a hydrogen atom, a C₁-C₆ alkyl, a C₂-C₆ alkenyl or a C₂-C₆ alkynyl group; R^(c) being a hydrogen atom, a C₁-C₆ alkyl, a C₂-C₆ alkenyl or a C₂-C₆ alkynyl group; R^(d) being a hydrogen atom, a C₁-C₆ alkyl, a C₂-C₆ alkenyl or a C₂-C₆ alkynyl group and Y^(a) being a direct bond, a C₁-C₆ alkylene, a C₂-C₆ alkenylene or a C₂-C₆ alkynylene group, wherein each heteroalkyl group contains at least one carbon atom and one or more hydrogen atoms may be replaced by fluorine or chlorine atoms.

Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, isopropyloxy, butoxy, tert-butyloxy, methoxymethyl, ethoxymethyl, —CH₂CH₂OH, —CH₂OH, methoxyethyl, 1-methoxyethyl, 1-ethoxyethyl, 2-methoxyethyl or 2-ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropylethylamino, methylamino methyl, ethylamino methyl, diisopropylamino ethyl, methylthio, ethylthio, isopropylthio, enol ether, dimethylamino methyl, dimethylamino ethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, propionyloxy, acetylamino or propionylamino, carboxymethyl, carboxyethyl or carboxypropyl, N-ethyl-N-methylcarbamoyl or N-methylcarbamoyl. Further examples of heteroalkyl groups are nitrile, isonitrile, cyanate, thio-cyanate, isocyanate, isothiocyanate and alkylnitrile groups.

The expression alkoxy refers to an alkyl group singular bonded to oxygen.

The expression alkylthio refers to an alkyl group singular bonded to sulfur.

The expressions cycloalkyl and carbocyclic ring refer to a saturated cyclic group of hydrocarbons that contains one or more rings, preferably 1 or 2), and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10, especially 3, 4, 5, 6 or 7 ring carbon atoms, e.g. a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, decalinyl, bicyclo-[4.3.0]nonyl, tetraline, or cyclopentylcyclohexyl group. The expression cycloalkyl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, ═O, SH, NH₂, ═NH, N₃ or NO₂ groups, thus, for example, cyclic ketones such as, for example, cyclohexanone, 2-cyclohexenone or cyclopentanone. Further specific examples of cycloalkyl groups are a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetraline, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl group.

The expression aryl refers to an aromatic group that contains one or more rings containing from 6 to 14 ring carbon atoms, preferably from 6 to 10, especially 6, ring carbon atoms.

The expression heteroaryl refers to an aromatic group that contains one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10, especially 5 or 6, ring atoms, and contains one or more, preferably 1, 2, 3 or 4, oxygen, nitrogen, phosphorus or sulfur ring atoms, preferably O, S or N. Examples are pyridyl (e.g. 4-pyridyl), imidazolyl (e.g. 2-imidazolyl), phenylpyrrolyl (e.g. 3-phenylpyrrolyl), thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3′-bifuryl, pyrazolyl (e.g. 3-pyrazolyl) and isoquinolinyl groups. The expression heterocycloalkyl refers to a cycloalkyl group as defined above in which one or more (preferably 1, 2 or 3) ring carbon atoms, each independently, have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom). A heterocycloalkyl group has preferably 1 or 2 ring(s) containing from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably selected from C, O, N and S). The expression heterocycloalkyl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, ═O, SH, ═S, NH₂, ═NH, N₃ or NO₂ groups. Examples are a piperidyl, prolinyl, imidazolidinyl, piperazinyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydro-thiophenyl, tetrahydropyranyl, tetrahydrofuryl or 2-pyrazolinyl group and also lactames, lactones, cyclic imides and cyclic anhydrides.

The expression alkylcycloalkyl refers to a group that contains both cycloalkyl and also alkyl, alkenyl or alkynyl groups in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl, alkenylcycloalkyl and alkynylcycloalkyl groups. An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two ring systems having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms, and one or two alkyl, alkenyl or alkynyl groups having 1 or 2 to 6 carbon atoms. The expression aralkyl refers to a group containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, an arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, arylcycloalkenyl, alkylaryl-cycloalkyl and alkylarylcycloalkenyl group. Specific examples of aralkyls are toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, 1H-indene, tetraline, dihydronaphthalene, indanone, phenylcyclopentyl, cumene, cyclohexylphenyl, fluorene and indane. An aralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms.

The expression heteroalkylcycloalkyl refers to alkylcycloalkyl groups as defined above in which one or more, preferably 1, 2 or 3, carbon atoms have been replaced independently of each other by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom). A heteroalkylcycloalkyl group preferably contains 1 or 2 ring systems having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups having from 1 or 2 to 6 carbon atoms. Examples of such groups are alkylhetero-cycloalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, the cyclic groups being saturated or mono-, di- or tri-unsaturated.

The expression heterocyclic ring refers to heteroaryl group as defined above as well as to a cycloalkyl group or carbocyclic ring as defined above in which one or more (preferably 1, 2 or 3) ring carbon atoms, each independently, have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom, preferably by an oxygen, sulfur or nitrogen atom. A heterocyclic ring has preferably 1 or 2 ring(s) containing from 3 to 10, especially 3, 4, 5, 6 or 7 ring atoms, preferably selected from C, O, N and S. Examples are a aziridinyl, oxiranyl, thiiranyl, oxaziridinyl, dioxiranyl, azetidinyl, oxetanyl, thietanyl, diazetidinyl, dioxetanyl, dithietanyl, pyrrolidinyl, tetrahydrofuranyl, thiolanyl, phospholanyl, silolanyl, azolyl, thiazolyl, isothiazolyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperazinyl, morpholinyl, thiopmorpholinyl, trioxanyl, azepanyl, oxepanyl, thiepanyl, homopiperazinyl, or urotropinyl group.

The expression heteroaralkyl refers to an aralkyl group as defined above in which one or more (preferably 1, 2, 3 or 4) carbon atoms, each independently, have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus, boron or sulfur atom (preferably oxygen, sulfur or nitrogen), that is to say to a group containing both aryl or heteroaryl, respectively, and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with the above definitions. A heteroaralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 5 or 6 to 10 ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms, wherein 1, 2, 3 or 4 of these carbon atoms have been replaced by oxygen, sulfur or nitrogen atoms.

Examples are arylheteroalkyl, arylheterocycloalkyl, arylheterocycloalkenyl, arylalkyl-heterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl, arylalkylheterocyclo-alkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroaryl-cycloalkyl, heteroarylcycloalkenyl, heteroarylheterocycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkylheterocycloalkenyl, heteroarylheteroalkylcycloalkyl, heteroarylheteroalkylcycloalkenyl and heteroarylheteroalkylheterocycloalkyl groups, the cyclic groups being saturated or mono-, di- or tri-unsaturated. Specific examples are a tetrahydroisoquinolinyl, benzoyl, 2- or 3-ethylindolyl, 4-methylpyridino, 2-, 3- or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or 4-carboxyphenylalkyl group.

As already stated above, the expressions cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl also refer to groups in which one or more hydrogen atoms of such groups have been replaced independently of each other by fluorine, chlorine, bromine or iodine atoms or by OH, ═O, SH, ═S, NH₂, ═NH, N₃ or NO₂ groups.

The general term ring as used herein, unless defined otherwise, includes cycloalkyl groups or carbocyclic rings, heterocyclic rings, aryl groups, and heteroaryl groups.

The expressions “halo”, “halogen” or “halogen atom” as used herein means fluorine, chlorine, bromine, or iodine, preferably fluorine and/or chlorine.

The expression mono- or disaccharide, and derivatives thereof as used herein means a carbohydrate or sugar belonging to or derived from the group of monosaccharides or disaccharides.

Examples of mono-, disaccharides, and derivatives include glucose, 3-O-methyl-glucose, 1-deoxy-glucose, 6-deoxy-glucose, galactose, mannose, fructose, xylose, ribose, cellobiose, maltose, lactose, gentiobiose, saccharose, trehalose and mannitol, sorbitol and ribitol. Preferably, the saccharides are D-form saccharides, e.g. D-glucose, 3-O-methyl-D-glucose, 1-deoxy-D-glucose, or 6-deoxy-D-glucose, D-galactose, D-mannose.

As used herein a wording defining the limits of a range of length such as, e. g., “from 1 to 5” means any integer from 1 to 5, i. e. 1, 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.

The expression “—C(═O)O-motif” is used herein in order to clearly define a group comprising an sp²-hybridized carbonyl carbon attached (i) to any carbon or hetero atom and (ii) to an oxygen which in turn can be attached to hydrogen or any other chemical atom. The term “carboxyl group” is avoided for the description of a “—C(═O)O-motif” because it could be mistaken as describing the carboxylic acid only.

The term “in alpha position” is used to describe a directly adjacent position, while the term “in beta position” indicates a neighboring position of an atom or group A and an atom or group B, characterized in that one further atom or group is localized between A and B.

As used herein, the term oxamide refers to the arbitrarily substituted organic compound comprising 2 carbonyl carbons and two nitrogens, which compound is an arbitrarily substituted diamide derived from any oxalic acid.

Those skilled in the art will readily recognize that some of the n-3 PUFA analogues of general formula (I) of the present invention represent “bioisosteres” of the naturally occurring epoxymetabolites produced by cytochrome P450 (CYP) enzymes from omega-3 (n-3) polyunsaturated fatty acids (PUFAs). A bioisostere is a compound resulting from the exchange of an atom or of a group of atoms with an alternative, broadly similar, atom or group of atoms, thereby creating a new compound with similar biological properties to the parent compound. Bioisosterism has, for example, been used by medicinal chemists for improving desired biological or physical properties of a compound, e.g. to attenuate toxicity, modify activity, alter pharmacokinetics and/or metabolism of a compound. For example, the replacement of a hydrogen atom with fluorine at a site of metabolic oxidation in a compound may prevent such metabolism from taking place. Because fluorine is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, said compound may have a longer half-life. Another example is the bioisosteric replacement of carboxylic acid groups which has resulted in analogues showing improved bioavailability, enhanced blood-brain barrier penetration, increased activity, better chemical stability and/or selectivity towards the target (see, e.g. the textbook “The practice of medicinal chemistry”, edited by Camille Georges Wermuth, 3^(rd) edition, Academic Press, 2008, e.g. p. 303-310; Ballatore C. et al. “Carboxylic Acid (Bio)Isosteres in Drug Design”, Chem Med Chem 8, 385-395 (2013)). Further, bioisosterism can also be used to provide a “prodrug” of a compound, i.e. a compound that is initially administered to a subject or patient in an inactive (or less active) form, and then becomes modified in vivo to its active form through the normal metabolic processes of the body. For example, conjugation of a compound with lipid and/or sugar units has resulted in analogues (prodrugs) showing increased drug delivery compared to the parent compound (see, e.g. Wong A. and Toth I. “Lipid, Sugar and Liposaccharide Based Delivery Systems”, Current Medicinal Chemistry 8, 1123-1136 (2001)).

The n-3 PUFA analogues of general formula (I) of the present invention can be prepared in a number of ways well known to one skilled in the art of organic synthesis. For example, the compounds of the present invention can be synthesized according to the general reaction schemes shown below using synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Unless indicated otherwise, all variables, e.g. n, k, R² (also referred to as R₂), R⁶, R⁷, R⁸, R⁴¹, R⁴², R⁴⁴ and R⁴⁵, have the above defined meaning. As starting materials reagents of standard commercial grade can be used without further purification, or can be readily prepared from such materials by routine methods. Those skilled in the art of organic synthesis will recognize that starting materials and reaction conditions may be varied including additional steps employed to produce compounds encompassed by the present invention

In a second aspect, the present invention is directed to a pharmaceutical composition comprising a compound according to formula (I) in combination with a physiologically acceptable excipient.

It is especially preferred to combine the preferred embodiments of the individual generic groups of formula (I) in any possible manner.

In a further aspect, the present invention is directed to a compound of formula (I) or a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of cardiovascular diseases, preferably selected from the group consisting of atrial fibrillation, ventricular arrhythmia, and heart failure.

In a further aspect, the present invention is directed to a compound of formula (I) or a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of atrial fibrillation, ventricul ararrhythmia, heart failure, coronary artery disease, myocardial infarction, maladaptive cardiac hypertrophy, and cardiac arrhythmias including ventricular extrasystoles, ventricular tachycardia, malignant ventricular tachycardia, atrial tachycardia, atrial flutter and atrial fibrillation, dilatative cardiomyopathy, and hypertensive heart disease, preferably selected from the group consisting of atrial fibrillation, atrial tachycardia, ventricular arrhythmia, heart failure, preferably selected from the group consisting of atrial fibrillation, atrial tachycardia, ventricular arrhythmia and heart failure.

In a preferred embodiment, the compound or composition for use according to the invention is administered orally, topically, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intranasally, preferably orally or intraveneously, more preferably orally.

It is further preferred that the compound or composition for use according to the invention is a dosage form selected from the group consisting of a spray, an aerosol, a foam, an inhalant, a powder, a tablet, a capsule, a soft gelatin capsule, a tea, a syrup, a granule, a chewable tablet, a salve, a cream, a gel, a suppository, a lozenge, a liposome composition and a solution suitable for injection.

The pharmaceutical compositions according to the present invention comprise at least one compound of formula (I) and, optionally, one or more carrier substances, e.g. cyclodextrins such as hydroxypropyl β-cyclodextrin, micelles or liposomes, excipients and/or adjuvants. Pharmaceutical compositions may additionally comprise, for example, one or more of water, buffers such as, e.g., neutral buffered saline or phosphate buffered saline, ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates such as e.g., glucose, mannose, sucrose or dextrans, mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives. Furthermore, one or more other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein. For instance, the compounds of the invention may advantageously be employed in combination with an antibiotic, anti-fungal, or anti-viral agent, an anti-histamine, a non-steroidal anti-inflammatory drug, a disease modifying anti-rheumatic drug, an anti-inflammatory drug to treat an autoimmune disease, a cytostatic drug, a drug with smooth muscle activity modulatory activity, an antihypertensive drug, a betablocker, an antiarrhythmic drug, a drug to treat heart failure, an antithrombotic drug, an antiplatelet drug, or mixtures of the aforementioned.

Preferably, the invention relates to a combination preparation or kit-of-parts comprising at least one compound according to the invention and at least one drug from the group comprising an antihypertensive drug, a betablocker, an antiarrhythmic drug, a drug to treat heart failure, an antithrombotic drug, an antiplatelet drug, an anti-rheumatic drug, and/or an anti-inflammatory drug to treat an autoimmune disease.

Pharmaceutical compositions may be formulated for any appropriate route of administration, including, for example, topical such as, e.g., transdermal or ocular, oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular such as, e.g., intravenous, intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate. Formulation for topical administration may be preferred for certain conditions such as, e.g., in the treatment of skin conditions such as burns or itch.

Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., corn starch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g., magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed. Methods for preparing such compositions are known (see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., Lea and Febiger (1990)).

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as, e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as, e.g., peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as, e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; and dispersing or wetting agents such as, e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil such as, e.g., arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as, e.g., olive oil or arachis oil, a mineral oil such as, e.g., liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as, e.g., gum acacia or gum tragacanth, naturally-occurring phosphatides such as, e.g., soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as, e.g., sorbitan monoleate, and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide such as, e.g., polyoxyethylene sorbitan monoleate. An emulsion may also comprise one or more sweetening and/or flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavoring agents and/or coloring agents.

Compounds may be formulated for local or topical administration, such as for topical application to the skin or mucous membranes, such as in the eye. Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components. Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include water; organic solvents such as alcohols such as, e.g., ethanol or isopropyl alcohol or glycerin; glycols such as, e.g., butylene, isoprene or propylene glycol; aliphatic alcohols such as, e.g., lanolin; mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin; lipid-based materials such as fatty acids, acylglycerols including oils, such as, e.g., mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes; protein-based materials such as collagen and gelatin; silicone-based materials, both non-volatile and volatile; and hydrocarbon-based materials such as microsponges and polymer matrices. A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale—The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

A topical formulation may be prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays, eye-drops and skin patches. The physical appearance and viscosity of such forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form; solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are often similar to one another, differing mainly in their viscosity; both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.

Suitable emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable viscosity adjusting agents include, but are not limited to, protective colloids or non-ionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within topical formulations.

Suitable preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils. Suitable fragrances and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in a topical formulation include, but are not limited to, abrasives, absorbents, anti-caking agents, anti-foaming agents, anti-static agents, astringents such as, e.g., witch hazel, alcohol and herbal extracts such as chamomile extract, binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.

An example of a suitable topical vehicle for formulation of a gel is: hydroxypropylcellulose (2.1%); 70/30 isopropyl alcohol/water (90.9%); propylene glycol (5.1%); and Polysorbate 80 (1.9%). An example of a suitable topical vehicle for formulation as a foam is: cetyl alcohol (1.1%); stearyl alcohol (0.5%); Quaternium 52 (1.0%); propylene glycol (2.0%); Ethanol 95 PGF3 (61.05%); deionized water (30.05%); P75 hydrocarbon propellant (4.30%). All percents are by weight.

Typical modes of delivery for topical compositions include application using the fingers; application using a physical applicator such as a cloth, tissue, swab, stick or brush; spraying including mist, aerosol or foam spraying; dropper application; sprinkling; soaking; and rinsing. Controlled release vehicles can also be used, and compositions may be formulated for transdermal administration as a transdermal patch.

A pharmaceutical composition may be formulated as inhaled formulations, including sprays, mists, or aerosols. Such formulations are particularly useful for the treatment of asthma or other respiratory conditions. For inhalation formulations, the compounds provided herein may be delivered via any inhalation methods known to those skilled in the art. Such inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable. Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the subject method typically include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.

Inhalant compositions may comprise liquid or powdered compositions containing the active ingredient that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent, e.g., isotonic saline or bacteriostatic water. The solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Formulations or compositions suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is administered, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable powder compositions include, by way of illustration, powdered preparations of the active ingredient thoroughly intermixed with lactose or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which may be inserted by the patient into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation.

Pharmaceutical compositions may also be prepared in the form of suppositories such as e.g., for rectal administration. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.

Pharmaceutical compositions may be formulated as sustained release formulations such as, i.e., a formulation such as a capsule that creates a slow release of modulator following administration. Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of modulator release. The amount of modulator contained within a sustained release formulation depends upon, for example, the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

For the treatment of cardiac damage, especially cardiac arrhythmias, the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements. Active compounds according to the present invention are generally administered in an effective amount, e.g., in a therapeutically effective amount. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body weight per day, about 0.5 mg to about 7 g per patient per day. The daily dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, i.e. other drugs being used to treat the patient, and the severity of the particular disease undergoing therapy.

Preferred compounds of the invention will have certain pharmacological properties. Such properties include, but are not limited to oral bioavailability, such that the preferred oral dosage forms discussed above can provide therapeutically effective levels of the compound in vivo.

Examples of conditions and diseases associated with cardiovascular diseases and which can be treated with the compounds for use according to the present invention include atrial fibrillation, ventricular arrhythmia, heart failure, coronary artery disease, myocardial infarction, maladaptive cardiac hypertrophy, and cardiac arrhythmias including ventricular extrasystoles, ventricular tachycardia, malignant ventricular tachycardia, atrial tachycardia, atrial flutter and atrial fibrillation, dilatative cardiomyopathy, and hypertensive heart disease, preferably selected from the group consisting of atrial fibrillation, atrial tachycardia, ventricular arrhythmia, heart failure.

n-3 PUFA derivatives provided herein are preferably administered to a patient such as, e.g., a human, orally or parenterally, and are present within at least one body fluid or tissue of the patient.

In a further aspect, the present invention is directed to a method for the treatment of a cardiovascular disease, preferably a cardiovascular disease as listed above, comprising the step of administering a compound or composition of the invention to a subject in need thereof, preferably a mammal, more preferably a human, in an effective amount.

As used herein, the term “treatment” encompasses both any type of disease-modifying treatment and including symptomatic treatment, i.e., a treatment after the onset of symptoms, either of which may be prophylactic, i.e., Disease However, disease-modifying treatment may involve administration before the onset of symptoms, in order to prevent, at least delay or reduce the severity of symptoms after onset, or. A disease-modifying treatment may also be therapeutic, i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms. A treatment after onset of symptoms may also simply involve stopping progressing of the disease (stable disease). In certain embodiment, the n-3 PUFA derivatives provided herein are administered prophylactically, i.e., before the onset of the disease and/or symptoms, ideally, but not necessarily, to actually prevent the diseases and/or symptoms. It is to be understood that the term prophylaxis and prophylactic in the context of the present invention, simply describes that the compound(s) of the present invention are administered before the onset of symptoms. A prophylactic administration may an administration before the onset of symptoms that are clearly associated with a disease discussed herein: the n-3 PUFA derivatives provided herein may, e.g., be administered to a subject prophylactically when he or she displays certain conditions that may indicate a propensity to develop one of the conditions or diseases that can be treated with one of the n-3 PUFA derivatives of the present invention. Such indicative conditions are, e.g. high blood pressure or diabetes. Such a prophylactic treatment is called primary prophylaxis. In another embodiment, the n-3 PUFA derivatives provided herein may be administered to a subject prophylactically when he or she has previously suffered from a condition or disease that can be treated with the n-3 PUFA derivatives of the present invention, but currently does not display any symptoms. Such a prophylactic treatment is called secondary prophylaxis. Patients receiving the n-3 PUFA derivatives for the purpose of primary or secondary prophylaxis are considered to be in need of such a treatment. Patients may include but are not limited to mammals, especially humans, domesticated companion animals such as dogs, cats, horses, and livestock such as cattle, pigs, sheep, with dosages as described herein.

As the person skilled in the art will appreciate, a wide variety of condition and diseases will benefit from the administration of the n-3PUFA derivatives of the present invention, the most prominent of which are cardiovascular diseases.

The activity of the n-3 PUFA analogues according to the invention can, for example, be determined in appropriate in vitro and/or in vivo assays. For instance, the biological activity of the n-3 PUFA analogues according to the present invention may be determined using the established cell model of Kang and Leaf (Proc Natl Acad Sci USA, 1994. 91(21): p. 9886-90.) known to those skilled in the art.

The following figures and examples serve to illustrate the invention and are not intended to limit the scope of the invention as described in the appended claims.

FIGURES

FIG. 1 is a column chart showing compounds 1 to 5 (Comp-01 to Comp-05), being examples of the invention, and other related structures (Comp-06 to Comp-13) with their potential to reduce spontaneous beating of NRCMs (For further details see Example 2 below).

FIG. 2 is a column chart showing a treatment with compound 2 (Comp-02), a synthetic agonist of 17,18-EEQ, which ameliorates the AF burden (A) and severity (B) of atrial fibrillation in a mouse model of moderate cardiac hypertrophy.

FIG. 3 is a column chart showing a treatment with compound 3 (Comp-03), a synthetic agonist of 17,18-EEQ, which ameliorates the duration (A) and severity (B) of cardiac arrhythmias in a rat model of myocardial infarction.

FIG. 4 is a column chart showing a treatment with compound 3 (Comp-03), a synthetic agonist of 17,18-EEQ, improving the post-ischemic functional recovery of isolated perfused mice hearts.

FIG. 5 is a column chart showing the inhibition of soluble epoxide hydrolase (sEH) by compounds of the invention (Comp-01 to Comp-03) in comparison to related analogs (Comp-07, Comp-08 and Comp-10).

FIG. 6 is a column chart showing the permeability potential of metabolically robust analogs of CYP-eicosanoids all part of the invention (Comp-01 to Comp-04) tested in Caco-2 cells.

FIG. 7 is a table summarizing data about the incorporation of compounds of the invention (Comp-02 and Comp-04) and other related compounds into membrane phospholipids.

FIG. 8 shows that continuous infusion of 100 nM of Comp-02 did not induce any obvious negative side effects. After global ischemia, contractility of the control hearts (n=5) was strongly reduced.

FIG. 9A to 9B show, that Comp-02 partially protected against OGD-induced damage in primary cardiomyocytes. FIG. 9C shows the LDH-release by Comp-02 and 17,18-EEQ.

EXAMPLE 1 SYNTHESIS OF COMPOUNDS

In the following the synthesis of selected compounds of the invention is illustrated.

Compound 1 (Comp-01)

Synthesis of compound 1 (Comp-01) was analogous to synthesis of compound 3 (Comp-03), while the urea-group was introduced following the synthetic route described in patent application WO2010/081683 (example 13).

Compound 2 (Comp-02)

Summary of Synthesis

General Method

NMR spectra were recorded on Bruker Avance 400 MHz for ¹HNMR and 100 MHz for ¹³CNMR. LCMS were taken on a quadrupole Mass Spectrometer on Shimadzu LCMS 2010 (Column: sepax ODS 50×2.0 mm, 5 um) or Agilent 1200 HPLC, 1956 MSD (Column: Shim-pack XR-ODS 30×3.0 mm, 2.2 um) operating in ES (+) ionization mode. Chromatographic purifications were by flash chromatography using 100˜200 mesh silica gel. Anhydrous solvents were pre-treated with 3A MS column before used. All commercially available reagents were used as received unless otherwise stated.

General Procedure for Preparation of Compound 2

Reagent MW. amount mmol ratio Other Info. Compound 1 136.53 100 g 732.44 1 MeNH₂—HCl 67.52 64.29 g 952.17 1.3 Et₃N 101.19 185.29 g 1830 2.5 THE 2 L Product 131.13 70 g 533.82 Yield: 73% (compound 2)

Methanamine (64.29 g, 952.17 mmol, 1.30 Eq) in 500 mL THF was added Et₃N (75 g, 732.44 mmol), the solution was added to Compound 1 (100.00 g, 732.44 mmol, 1.00 eq), Et₃N (111 g, 1.1 mol) in THF (1.5 L) at −10° C. And the mixture was stirred at 25° C. for 16 h. Then the mixture was filtered, the filtrate was washed with 2N HCl (500 mL), extracted with EA (300 mL*4), concentrated and purified by silica gel (PE:EA=3:1 to 1:1) to afford Compound 2 (70.00 g, 533.82 mmol, 72.88% yield) as a yellow oil.

TLC Information (PE:EtOAc=2:1); R_(f) (Comp-02)=0.39; LCMS: ET2662-1-P1A (M+H⁺): 131.7; ¹H NMR (CDCl₃, 400 MHz) 4.36˜4.24 (q, J=8 Hz, 2H), 2.93˜2.85 (d, J=4 Hz, 3H), 1.38˜1.30 (t, J=8 Hz, 3H)

General Procedure for Preparation of Compound 4

Reagent MW. amount mmol ratio Other Info. Compound 3 98.14 47.5 g 484 1 Compound 4 147.13- 78.33 g 532.4 1.1 PPh₃ 262.29 133.3 g 508.2 1.05 DIAD 202.21 107.66 g 532.4 1.1 THF 1.8 L Product 227.26 42.5 g 374.02 Yield 77.3% (compound 5)

A solution of Compound 3 (47.50 g, 484.00 mmol, 1.00 eq.) and DIAD (107.66 g, 532.40 mmol, 1.10 eq.) in anhydrous THF (50 mL) was slowly added via cannula to a 0° C. solution of Compound 4 (78.33 g, 532.40 mmol, 1.10 eq.) and PPh₃ (133.30 g, 508.20 mmol, 1.05 eq.) in anhydrous THF (100 mL). The flask and cannula were washed with an additional portion of dry THF (30 mL) to ensure complete addition. The reaction was allowed to gradually warm to 25° C. and stirred for 18 h. Then H₂O (1000 mL) was added, extracted with EA (500 mL*2), concentrated and purified by silica gel (PE:EA=0-10:1) to give Compound 5 (42.5 g, 374.02 mmol, 77.28% yield) as a white solid.

TLC Information (PE:EtOAc=5:1); R_(f) (Comp-03)=0.2; R_(f) (Comp-05)=0.5; ¹H NMR (CDCl₃, 400 MHz) 7.86˜7.79 (m, 2H), 7.72˜7.67 (m, 2H), 3.73˜3.66 (t, J=8 Hz, 2H), 2.27˜2.20 (m, 2H), 1.95˜1.91 (t, J=4 Hz, 1H), 1.85˜1.75 (m, 2H), 1.61˜1.52 (m, 2H)

General Procedure for Preparation of Compound 6

Reagent MW. amount mmol ratio Other Info. Compound 5 227.26 88 g 387.22 1 NIS 224.98 130.68 g 580.83 1.5 AgNO₃ 169.87 16.44 g 96.81 0.25 THF 1.6 L Product (Compound 6) 353.15 118.6 g 335.8 Yield: 86%

NIS (130.68 g, 580.83 mmol, 1.50 eq.) was added in one portion to a solution of Compound 5 (88.00 g, 387.22 mmol, 1.00 eq.) and AgNO₃ (16.44 g, 96.81 mmol, 0.25 eq.) in anhydrous THF (1600 mL) at 25° C. The reaction head space was flushed with N₂ and the reaction mixture was protected from light with an aluminum foil wrap and stirred for 16 h. The mixture was poured into water (1000 mL), extracted with EA (600 mL*3), concentrated and purified by silica (PE:EA=10:1 to 2:1) to give Compound 6 (118.6 g, 1.01 mol, 86.78% yield) as a white solid.

TLC Information (PE:EtOAc=20:1); R_(f) (Comp-05)=0.22; R_(f) (Cpd 6)=0.21; ¹H NMR (CDCl₃, 400 MHz) 7.87˜7.82 (m, 2H), 7.74˜7.69 (m, 2H), 3.74˜3.67 (t, J=8 Hz, 2H), 2.45˜2.39 (t, J=8 Hz, 2H), 1.84˜1.74 (m, 2H), 1.61˜1.52 (m, 2H)

General Procedure for Preparation of Compound 7

Reagent MW. amount mmol ratio Other Info. Compound 6 353.15 157 g 444.57 1 BH_(3.)DMS 58 mL 577.94 1.3 2-methyl-2-butene 70.13 87.3 g 1240 2.8 AcOH 260 mL THF 1.2 L Product (compound 5) 355.17 135 g 380.1 Yield: 85%

2-methylbut-2-ene (87.30 g, 1.24 mol, 2.80 eq.) was added over 30 min to a 0° C. solution of BH3.Me2S (43.91 g, 577.94 mmol, 1.30 eq.) in THF (300 mL). After 1 h, the reaction mixture was warmed to 25° C. and stirred for 90 min. After re-cooling to 0° C., a solution of Compound 6 (157.00 g, 444.57 mmol, 1.00 eq.) in THF (900 mL) was added slowly over 1 h. Upon complete addition, the cold bath was removed and the reaction mixture was stirred at 25° C. After 2 h, the reaction was cooled again to 0° C. where upon glacial AcOH (260 mL) was added slowly over 30 min (Caution: gas evolution) and stirred at 25° C. for 16 h. TLC (PE:EA=10:1) show the reaction was completed, the mixture was pour into water (1 L), extracted with EA (300 mL*2), concentrated and purified by silica gel (PE:EA=0-10:1) to give Compound 7 (135 g, 380.1 mmol, 85.50% yield) as a yellow oil.

TLC Information (PE:EtOAc=10:1); R_(f) (Cpd 6)=0.5; R_(f) (Cpd 7)=0.55; ¹H NMR: (CDCl₃, 400 MHz) 7.88˜7.80 (m, 2H), 7.75˜7.67 (m, 2H), 6.24˜6.11 (m, 2H), 3.74˜3.66 (t, J=8 Hz, 2H), 2.24˜2.15 (q, J=8 Hz, 2H), 1.78˜1.67 (m, 2H), 1.55˜1.44 (m, 2H)

General Procedure for Preparation of Compound 8

Reagent MW. amount mmol ratio Other Info. Compound 7 355.17 138 g 388.55 1 N₂H₄•H₂O 50.06 97.25 g 1940 5 MeOH 2 L Product (compound 8) 225.07 81 g 683.79 Yield: 92%

N₂H₄.H₂O (97.25 g, 1.94 mol, 5.00 eq.) was added to a solution of Compound 7 (138.00 g, 388.55 mmol, 1.00 eq) in anhydrous MeOH (2.00 L) at 0° C. and stirred at 25° C. for 18 h, TLC (PE:EA=10:1) show the reaction was completed, the reaction mixture was concentrated, the residue was poured into DCM (5000 mL) and stirred for 30 mins. Filtered and the filter cake was washed with DCM (1 L*2), the filtrate was concentrated to give Compound 8 (162.00 g, crude) as a yellow oil.

TLC Information (PE:EtOAc=10:1); R_(f) (Cpd 7)=0.5; R_(f) (Cpd 8)=0; TLC Information (DCM: MeOH=10:1); R_(f) (Cpd 7)=1; R_(f) (Cpd 8)=0.2; ¹H NMR: (CDCl₃, 400 MHz) 6.19˜6.07 (m, 2H), 2.73˜2.59 (m, 2H), 2.20˜2.05 (m, 2H), 1.75˜1.55 (m, 2H), 1.51˜1.36 (m, 4H)

General Procedure for Preparation of Compound 9

Reagent MW. amount mmol ratio Other Info. Compound 8 225.07 92 g 408.76 1 Compound 2 131.13 53.6 g 408.76 1 Et3N 101.19 49.64 g 590.51 1.2 EtOH 1.5 L Product (compound 9) 310.13 90 g 232.16 Yield: 57%

Compound 8 (92.00 g, 408.76 mmol, 1.00 eq) Compound 2 (53.60 g, 408.76 mmol, 1.00 eq) and Et₃N (49.64 g, 490.51 mmol, 1.20 eq) in anhydrous ethanol (1.5 L) was heated at 60° C. for 20 h. TLC (DCM:MeOH=10:1) show the reaction was completed, the mixture was concentrated to about 300 mL. Filtered and concentrated to give Compound 9 (90 g, 232.16 mmol, 57% yield) as a white solid.

TLC Information (DCM: MeOH=10:1); R_(f) (Cpd 8)=0.2; R_(f) (Cpd 9)=0.5; ¹H NMR: (CDCl₃, 400 MHz) 7.57˜7.37 (s, 2H), 6.25˜6.20 (d, J=8 Hz, 1H), 6.18˜6.11 (q, J=8 Hz, 1H), 3.37-3.30 (q, J=8 Hz, 2H), 2.93˜2.88 (d, J=4 Hz, 3H), 2.21˜2.13 (m, 2H), 1.66˜1.56 (m, 2H), 1.53˜1.43 (m, 2H)

General Procedure for Preparation of Compound 12

Reagent MW. amount mmol ratio Other Info. Compound 10 114.1 25 g 197.2 1 90% Compound 11 114.18 27.02 g 236.63 1.2 In(OTf)₃ 560 22.09 g 39.44 0.2 toluene 350 mL Compound 12 200.27 35 g 139.81 Yield: 71%

Compound 10 (25.00 g, 197.20 mmol, 1.00 eq.) in toluene (75 mL) was added to a solution of Compound 11 (27.02 g, 236.63 mmol, 1.20 eq.) In (OTf)₃ (22.09 g, 39.44 mmol, 0.20 eq) in toluene (275 mL) over 20 mins. Then the mixture was stirred at 25° C. for 48 h. The mixture was concentrated and purified by silica gel (PE:EA=20:1) to give ethyl Compound 12 (35.00 g, 139.81 mmol, 70.90% yield, 80% purity) as a yellow oil.

TLC Information (PE:EtOAc=10:1); R_(f) (Cpd 11)=0.21; R_(f) (Cpd 12)=0.55; ¹H NMR: (CDCl₃, 400 MHz) 5.86˜5.72 (m, 1H), 5.03˜5.86 (m, 2H), 4.24˜4.17 (q, J=8 Hz, 2H), 4.07˜4.01 (s, 2H), 3.54˜3.47 (t, J=8 Hz, 2H), 2.09˜1.98 (m, 2H), 1.68˜1.55 (m, 2H), 1.45˜1.32 (m, 4H), 1.30˜1.25 (t, J=8 Hz, 3H)

General Procedure for Preparation of Compound 13

Reagent MW. amount mmol ratio Other Info. Compound 12 200.27 10.07 g 50.3 1.2 90% Compound 9 310.13 13 g 41.92 1 9-BBN 100.6 mL 100.6 2.4 Na₂CO₃ 200 mL 2M Pd(PPh₃)Cl₂ 701.9 1.47 g 2.1 0.05 THF 800 mL Compound 13 384.51 6.5 g 16.06 Yield: 38%

To an oven-dried flask containing 9-BBN (17.53 g, 100.60 mmol, 2.40 eq) in THF (540 mL) was added a solution of Compound 12 (10.07 g, 50.30 mmol, 1.20 eq) in THF (60 mL) at 0° C. After stirring at 25° C. for 16 h, an aqueous solution of Na₂CO₃ (200 mL of 2 M soln prepared from argon sparged H₂O) was added. After 2 h, Pd(PPh₃)₂Cl₂ (1.47 g, 2.10 mmol, 0.05 eq) was added followed by Compound 9 (13.00 g, 41.92 mmol, 1.00 eq) dissolved in THF (200 mL). The resulting red solution was protected from light. The reaction was stirred at 50° C. for 5 h. LCMS show the reaction was completed. After cooling to 25° C., the reaction mixture was concentrated in vacuo and the residue was purified by silica gel (PE:EA=10:1 to 3:1) to give Compound 13 (6.5 g, 16.06 mmol, 38.31% yield, 95% purity) as a yellow solid.

TLC Information (PE:EtOAc=2:1); R_(f) (Cpd 12)=0.3; R_(f) (Cpd 13)=0.3; LCMS: ET2662-38-P1D (M+H⁺): 385.1; ¹H NMR: (CDCl₃, 400 MHz) 7.57˜7.38 (s, 1H), 5.41˜5.25 (m, 2H), 4.25˜4.17 (q, J=8 Hz, 2H), 4.07˜4.02 (s, 2H), 3.54˜3.47 (t, J=8 Hz, 2H), 3.34˜3.26 (q, J=8 Hz, 2H), 2.92˜2.87 (d, J=8 Hz, 3H), 2.08˜1.94 (m, 4H), 1.65˜1.51 (m, 4H), 1.43˜1.23 (m, 13H)

General Procedure for Preparation of Comp-02

Reagent MW. amount mmol ratio Other Info. Compound 13 384.51 7.5 g 19.51 1 90% LiOH 23.95 0.9341 g 39.02 2 H₂O 40 mL THF 70 mL Compound 13 356.46 4 g 10.72 Yield: 55%

To a solution of Compound 13 (7.50 g, 19.51 mmol, 1.00 eq.) in THF (70.00 mL) was added LiOH (934.31 mg, 39.02 mmol, 2.00 eq.) in H₂O (40.00 mL) at 0° C. and then the reaction mixture was stirred at 0-25° C. for 1 h. LCMS show the reaction was completed. Then H₂O (60 mL) was added to the reaction mixture, the aqueous phase was treated with 3 N HCl (10 mL) to pH=3-4, extracted with EA (100 mL*3), dried, the organic phase was concentrated to give crude product. The residue was purified by column on gel (PE:EA=5:1 to EA) to give Comp-02 (4.00 g, 10.72 mmol, 54.95% yield, 95.51% purity)

TLC Information (DCM: MeOH=10:1); R_(f) (Cpd 13)=0.9; R_(f) (Comp-02)=0.4; MS: ET2662-43-P1C (M+Na⁺): 379.2; ¹H NMR (CDCl₃, 400 MHz) 7.84 (s, 1H), 7.74 (s, 1H), 5.40˜5.32 (m, 2H), 4.11 (s, 2H), 3.59˜3.55 (t, J=6.4 Hz, 2H), 3.35˜3.32 (t, J=6.8 Hz, 2H), 2.92˜2.91 (d, J=5.2 Hz, 3H), 2.07˜2.00 (m, 4H), 1.64-1.59 (m, 4H), 1.42˜1.32 (m, 10H); ¹³C NMR (CDCl3, 100 MHz) δ 173.7, 160.7, 159.8, 130.5, 129.0, 72.0, 67.8, 39.7, 29.4, 29.3, 29.0, 29.0, 28.6, 27.1, 26.8, 26.7, 25.8

Compound 3 (Comp-03)

Summary of Synthesis

Synthesis of 2-(hex-5-yn-1-yl)isoindoline-1,3-dione (2)

Following literature precedent,¹ a solution of 5-hexyn-1-ol (1) (5 g, 1 equiv) and diisopropyl azodicarboxylate (DIAD, 10.5 g, 1.02 equiv) in anhydrous THF (30 mL) was slowly added via cannula to a 0° C. solution of phthalimide (7.5 g, 51 mmol) and triphenylphosphine (TPP, 13.4 g, 1 equiv) in anhydrous THF (50 mL). The flask and cannula were washed with an additional portion of dry THF (20 mL) to ensure complete addition. The reaction was allowed to gradually warm to room temperature overnight. After a total of 18 h, all volatiles were evaporated and the residue was purified using a Teledyne Isco Combiflash® RF chromatographic system (80 g SiO₂ column eluted with hexanes, 2 min; 0-20% EtOAc/hexanes, 12 min; 20% EtOAc/hexanes, 6 min) to give 2 (8.3 g, 72%) as a white solid whose spectral values were identical to those reported.²

Synthesis of 2-(6-iodohex-5-yn-1-yl)isoindoline-1,3-dione (3)

Following literature precedent,³ N-iodosuccinimide (NIS, 7.4 g, 1.5 equiv) was added in one portion to a rt solution of alkyne 2 (5.0 g, 22 mmol) and AgNO₃ (0.93 mg, 0.25 equiv) in anhydrous THF (120 mL). The reaction head space was flushed with argon and the reaction mixture was protected from light with an aluminum foil wrap. After 4 h, the reaction mixture was poured into H₂O (200 mL) and extracted with Et₂O (2×50 mL). The ethereal extracts were washed with brine (3×60 mL) (Note: biphasic mixture turned brown). The combined aqueous phases were re-extracted with Et₂O (2×50 mL). The combined ethereal extracts were dried over Na₂SO₄, filtered, and concentrated on a rotary evaporator. The residue was purified using a Teledyne Isco Combiflash® RF chromatographic system (80 g SiO₂ column eluted with hexanes, 2 min; 0-40% EtOAc/hexanes, 8 min; 40% EtOAc/hexanes, 10 min; 40-100% EtOAc/hexanes, 5 min; 100%, EtOAc, 3 min) to give 3 (97%) as a white solid, mp 132.5-132.7° C. ¹H NMR (500 MHz, CDCl₃) δ 7.85 (ddd, J=5.4, 3.0, 1.0 Hz, 2H), 7.72 (ddd, J=5.5, 3.0, 1.0 Hz, 2H), 3.71 (t, J=7.1 Hz, 2H), 2.42 (t, J=7.0 Hz, 2H), 1.83-1.73 (m, 2H), 1.61-1.51 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 168.62, 134.14, 132.30, 123.44, 94.04, 37.60, 27.91, 25.89, 20.60, −6.27.

Synthesis of 2-(6-iodohex-5(Z)-en-1-yl)isoindoline-1,3-dione (4)

Following literature precedent,⁴ neat 2-methyl-2-butene (4.2 mL, 2.8 equiv) was added over 5 min to a 0° C. solution of BH₃.Me₂S (2.0 M in THF, 9.2 mL, 1.3 equiv) in THF (3 mL). After 1 h, the reaction mixture was warmed to room temperature and stirred for 90 min. After re-cooling to 0° C., a solution of iodoalkyne 3 (5 g, 1 equiv) in THF (30 mL) was added slowly over 5 min. Upon complete addition, the cold bath was removed and the reaction mixture was stirred at rt. After 2 h, the reaction was cooled again to 0° C. whereupon glacial AcOH (8.5 mL) was added slowly over 5 min (Caution: gas evolution). After stirring overnight (14 h), the reaction mixture was diluted with H₂O (20 mL), then carefully poured into a stirring, saturated sodium bicarbonate solution (40 mL). The biphasic mixture was extracted with ether (2×40 mL) and the combined ethereal extracts were washed with water, brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using a Teledyne Isco Combiflash® RF chromatographic system (40 g SiO₂ column eluted with 0-20% EtOAc/hexanes, 8 min; 20% EtOAc/hexanes, 6 min) to give a mixture (4.52 g) of 4 and borane side-product. Further purification was postponed until the next step.

Synthesis of 6-iodohex-5(Z)-en-1-amine (5)

Following literature precedent,⁵ 40% wt MeNH₂ in H₂O (15 mL) was added to a rt solution of crude 4 (4.52 g) in anhydrous EtOH (20 mL). After stirring overnight (18 h), the reaction mixture was poured into ice water (100 mL) and extracted with Et₂O (30 mL×2). The combined ethereal extracts were washed with cold 1N HCl solution (20 mL×2). The combined aqueous washes were adjusted to pH 8 with dilute, aq. NaOH. The solution was extracted with Et₂O (30 mL×2), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to give crude 5 (1.12 g) as a brown oil that was used in the next step without further purification. ¹H NMR (500 MHz, CDCl₃) δ 6.29-6.08 (m, 2H), 2.71 (tt, J=7.0, 1.8 Hz, 2H), 2.16 (app q, J=6.5 Hz, 2H), 1.78-1.52 (m, 2H).

Synthesis of N¹-(6-iodohex-5(Z)-en-1-yl)-N²-methyloxalamide (7)

Following literature precedent,⁶ a solution of iodoalkene 5 (1.12 g, 4.98 mmol), ethyl 2-(methylamino)-2-oxoacetate (6) (0.62 g, 1.2 equiv) and triethylamine (0.83 mL, 1.2 equiv) in anhydrous ethanol (10 mL) was heated at 60° C. After 20 h, the brown solution was cooled to rt and concentrated in vacuo. Purification of the residue using a Teledyne Isco Combiflash® RF chromatographic system (25 g SiO₂ column eluted with 0-50% EtOAc/hexanes, 10 min; 50% EtOAc/hexanes, 10 min) gave 7 (0.93 g, 60%) as a white solid, 99.7-99.8° C. ¹H NMR (500 MHz, CDCl₃) δ 7.46 (br s, 2H), 6.32-6.02 (m, 2H), 3.34 (app q, J=6.9 Hz, 2H), 2.91 (d, J=5.3 Hz, 3H), 2.18 (dt, J=7.5, 7.0 Hz, 2H), 1.68-1.59 (m, 2H), 1.54-1.42 (m, 2H);¹³C NMR (100 MHz, CDCl₃) δ 160.47, 159.70, 140.43, 83.07, 39.40, 34.11, 28.61, 26.15, 25.11.

Synthesis of ethyl 2-(oct-7-en-1-yloxy)acetate (10)

Following literature precedent,⁷ neat 8 (1.92 g, 1.2 equiv) was added to a rt suspension of In(OTf)₃ (1.57 g, 20 mol %) in anhydrous toluene (20 mL). Ethyl diazoacetate (9) (1.60 g, 14 mmol) was added slowly under an argon atmosphere over 5 min (caution: exothermic) to give a yellow solution. After 2 days, the reaction mixture was concentrated in vacuo and the residue was purified using a Teledyne Isco Combiflash® RF chromatographic system (25 g SiO₂ column eluted with 0-10% EtOAc/hexanes, 5 min; 10% EtOAc/hexanes, 8 min) to give 10 (2.72 g, 97%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 5.80 (ddt, J=16.9, 10.2, 6.6 Hz, 1H), 5.08-4.84 (m, 2H), 4.22 (q, J=7.1 Hz, 2H), 4.06 (s, 2H), 3.52 (t, J=6.7 Hz, 2H), 2.13-1.96 (m, 2H), 1.72-1.52 (m, 2H), 1.48-1.33 (m, 4H), 1.28 (t, J=7.1 Hz, 3H);¹³C NMR (100 MHz, CDCl₃) δ 170.70, 138.99, 114.48, 71.97, 68.48, 60.86, 33.84, 29.55, 28.84, 25.63, 14.34.

Synthesis of ethyl 2-((13-(2-(methylamino)-2-oxoacetamido)tridec-8(Z)-en-1-yl)oxy)acetate (11)

To an oven-dried flask containing ethyl 2-(oct-7-en-1-yloxy)acetate (10) (220 mg, 1.2 equiv) was added a solution of 9-BBN (0.5 M in THF, 2.4 equiv, 4.40 mL). After stirring at rt for 3 h, an aqueous solution of Na₂CO₃ (1.5 mL of 2 M soln prepared from argon sparged H₂O) was added. After 5 min, Pd(PPh₃)₂Cl₂ (33 mg, 5 mol %) was added followed by 7 (284 mg, 0.92 mmol) dissolved in THF (4 mL). The resulting red solution was protected from light while another portion of aq. Na₂CO₃ (0.5 mL of 2 M soln) was added. The reaction was continued overnight (14 h) at rt, then at 50° C. for 4 h. After cooling to rt, the reaction mixture was concentrated in vacuo and the residue was purified using a Teledyne Isco Combiflash® RF chromatographic system (24 g SiO₂ column eluted with 0-40% EtOAc/hexanes, 6 min; 40% EtOAc/hexanes, 8 min; 40-100% EtOAc/hexanes, 4 min) to give ether 11 (330 mg, 90%) as an off-white solid. An analytical sample was purified by preparative TLC to give 11 as a white low melting solid.

TLC: 50% EtOAc/hexanes, R_(f)˜0.49. ¹H NMR (500 MHz, CDCl₃) δ 7.45 (br s, 2H), 5.42-5.26 (m, 2H), 4.22 (q, J=7.2 Hz, 2H), 4.06 (s, 2H), 3.52 (t, J=6.7 Hz, 2H), 3.31 (dt, J=7.0, 6.5 Hz, 2H), 2.91 (d, J=5.1 Hz, 3H), 2.15-1.91 (m, 4H), 1.70-1.50 (m, 2H), 1.44-1.31 (m, 12H), 1.29 (t, J=7.1 Hz, 3H);¹³C NMR (100 MHz, CDCl₃) δ 170.62, 160.55, 159.66, 130.58, 128.86, 71.96, 68.64, 39.55, 29.61, 29.51, 29.30, 29.19, 27.20, 26.83, 26.67, 26.15, 25.93, 14.21.

Synthesis of 2-((13-(2-(methylamino)-2-oxoacetamido)tridec-8(Z)-en-1-yl)oxy)acetic acid (12)

To a rt solution of 11 (720 mg, 1.87 mmol) in THF (44 mL) was added LiOH (9 mL of 1.0 M aq. solution). After 48 h, the reaction was cooled to 4° C. and acidified to pH 4 using aq. 2 N HCl. The mixture was diluted with H₂O (10 mL) and extracted with EtOAc (15 mL×3). The combined organic extracts were dried over Na₂SO₄, filtered through a fritted funnel, and concentrated in vacuo. The crude material was purified using a Teledyne Isco Combiflash® RF chromatographic system (12 g SiO₂ column eluted with 0-80% EtOAc/hexanes, 15 min; 80% EtOAc/hexanes, 5 min) to give 12 (232 mg, 33%) as a white solid, mp 94.6-94.7° C.

¹H NMR (500 MHz, CDCl₃) δ 7.90 (s, 1H), 7.66 (s, 1H), 5.48-5.22 (m, 2H), 4.10 (s, 2H), 3.58 (t, J=6.5 Hz, 2H), 3.32 (dt, J=7.0, 6.5 Hz, 2H), 2.91 (d, J=5.2 Hz, 3H), 2.16-1.90 (m, 4H), 1.71-1.48 (m, 4H), 1.45-1.18 (m, 10H); ¹³C NMR (75 MHz, CD₃OD) δ 176.96, 160.32, 160.12, 130.65, 129.99, 72.51, 69.84, 39.45, 29.82, 29.58, 29.15, 27.71, 27.38, 27.24, 27.08, 26.83, 25.84, 25.03.

Synthesis of Comp-03:

A mixture of EDCI (275 mg, 1.3 equiv) and triethyleneglycol (1.5 mL, 10 equiv) was dried under high vacuum for 90 min. The reaction flask was flushed with argon and DMAP (175 mg, 1.3 equiv), acetonitrile (50 mL), and acid 12 (395 mg, 1.1 mmol) dissolved in CH₂Cl₂ (20 mL) were added. After 3 days, the reaction mixture was concentrated in vacuo, the crude residue was dissolved in EtOAc (20 mL) and washed with 1N HCl (20 mL) and brine (20 mL). The aqueous washings were re-extracted with EtOAc (20 mL×2). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified using a Teledyne Isco Combiflash® RF chromatographic system (12 g SiO₂ column eluted with 0-80% EtOAc/hexanes, 8 min; 80% EtOAc/hexanes, 4 min; 80-100% EtOAc/hexanes, 3 min; 100% EtOAc, 15 min; 10% MeOH/CH₂Cl₂, 5 min) to give analog 13 (174 mg, 32%) as a white solid, mp 65.3-65.8° C.

¹H NMR (500 MHz, CDCl₃) δ 7.46 (s, 2H), 5.41-5.27 (m, 2H), 4.33 (t, J=4.7 Hz, 2H), 4.11 (s, 2H), 3.77-3.70 (m, 4H), 3.70-3.64 (m, 4H), 3.61 (app t, J=4.5 Hz, 2H), 3.52 (t, J=6.7 Hz, 2H), 3.42 (t, J=6.1 Hz, OH), 3.31 (dt, J=7.0, 6.5 Hz, 2H), 2.91 (d, J=5.2 Hz, 3H), 2.44 (s, 1H), 2.05 (dt, J=7.5, 7.0 Hz, 2H), 2.00 (dt, J=7.0, 6.5 Hz, 2H), 1.62-1.50 (m, 4H), 1.45-1.21 (m, 10H); ¹³C NMR (125 MHz, CDCl₃) δ 170.86, 160.83, 159.95, 130.76, 129.12, 72.76, 72.21, 70.77, 70.52, 69.19, 68.34, 63.84, 61.92, 39.78, 29.84, 29.73, 29.54, 29.42, 29.02, 27.44, 27.09, 26.92, 26.42, 26.15.

Compound 4 (Comp-04)

Synthesis of Compound 4 (Comp-04) was analogous to synthesis of compound 2 (Comp-02), while the urea-group was introduced following the synthetic route described in patent application WO2010/081683 (example 6).

Compound 5 (Comp-05)

Summary of Synthesis

General Procedure for Preparation of Compound 4-2

Reagent MW. amount mol ratio Other Info. Cpd. 4-1 219.02 30.0 g  137 mmol  1.0 eq. Cpd. 1 98.14 13.4 g  137 mmol  1.0 eq. CuI 190.45 522 mg 2.74 mmol 0.02 eq. Pd(PPh₃)₄ 1155.56 1.58 g 1.37 mmol 0.01 eq. TEA 480 mL Product: (Cpd. 4-2) 189.25 21.0 g 99.9 mmol Yield: 73%

A mixture of Cpd.4-1 (30.0 g, 137 mmol, 1.0 eq) in TEA (480 mL) was added Cpd.1 (13.4 g, 137 mmol, 1.0 eq), CuI (522 mg, 2.74 mmol, 0.02 eq), Pd(PPh₃)₄ (1.58 g, 1.37 mmol, 0.01 eq) under N₂ at 25° C. and stirred at 25° C. for 16 hrs. TLC (petroleum ether/ethyl acetate=1/1, R_(f)=0.5) showed that the reaction was complete. The solution was poured into aq.NH₄Cl (1.0 L), extracted with DCM (200 mL*5), the combined organic layers were washed with brine, dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluted with petroleum ether: EtOAc (10:1, 1:1) to give Cpd.4-2 (21.0 g, 73% yield) as yellow oil.

¹H NMR: ET5008-6-P1b1 400 MHz CDCl₃; 7.30-7.24 (m, 1H), 7.10 (t, J=7.6 Hz, 1H), 6.73-6.65 (m, 2H), 4.19 (br, 2H), 3.74 (m, 2H), 2.54 (t, J=6.0 Hz, 2H), 1.87-1.68 (m, 4H), 1.50-1.45 (m, 1H).

General Procedure for Preparation of Cpd.4-3

Reagent MW. amount mol ratio Other Info. Cpd. 4-2 189.25 21.0 g  111 mmol 1.0 eq. Pd/C 500 mg MeOH 500 mL Product: (Cpd. 4-3) 193.28 17.0 g 83.6 mmol Yield: 75%

A mixture of Cpd.4-2 (21.0 g, 111 mmol, 1.0 eq) in MeOH (500 mL) was added Pd/C (500 mg) and stirred at 25° C. under 50 psi of H₂ for 16 hrs. LC-MS (ET5008-10-P1A5, product: RT=1.10 min) show that the reaction was complete. Then the solution was filtered and concentrated to give Cpd.4-3 (17.0 g, 75% yield) as yellow oil.

¹H NMR: ET5008-10-P1b1 400 MHz CDCl₃; 7.08-7.03 (m, 2H), 6.78-6.69 (m, 2H), 3.69-3.62 (m, 4H), 2.52 (t, J=8.0 Hz, 2H), 1.68-1.59 (m, 4H), 1.47-1.42 (m, 4H), 1.31-1.27 (m, 1H).

General Procedure for Preparation of Cpd.4-4

Reagent MW. amount mol ratio Other Info. Cpd. 4-3 193.28 17.0 g 88.0 mmol 1.0 eq. NaNO₂ 69.00 6.07 g 88.0 mmol 1.0 eq. KI 166.00 43.8 g  264 mmol 3.0 eq. Con•H₂SO₄ 98.08 30.2 g  308 mmol 3.5 eq. H₂O 560 mL Product: (Cpd. 4-4) 304.17 17.0 g 50.3 mmol Yield: 57%

Con.H₂SO₄ (30.2 g, 308 mmol, 3.5 eq) was added to Cpd.4-3 (17.0 g, 88.0 mmol, 1.0 eq) in H₂O (500 mL) at 0° C. under N₂. A solution of NaNO₂ (6.07 g, 88.0 mmol, 1.0 eq) in H₂O (30.0 mL) was added to the solution at 0° C. and stirred at 0° C. for 15 mins. A solution of KI (43.8 g, 264 mmol, 3.0 eq) in H₂O (30.0 mL) was added at 0° C. and the resulting suspension was warmed to 25° C. and stirred for 45 mins. TLC (petroleum ether/ethyl acetate=1/1, R_(f)=0.9) showed that the reaction was complete. H₂O (400 mL) was added, extracted with EtOAc (350 mL*3), the combined organic layers were washed with brine, dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluted with petroleum ether: EtOAc (100:1, 10:1) to give Cpd.4-4 (17.0 g, 57% yield) as brown oil.

¹H NMR: ET5008-22-P1b1 400 MHz CDCl₃; 7.80 (d, J=7.2 Hz, 1H), 7.28-7.23 (m, 1H), 7.21-7.18 (m, 1H), 6.89-6.85 (m, 1H), 3.65 (t, J=6.8 Hz, 2H), 2.71 (t, J=8.0 Hz, 2H), 1.61-1.50 (m, 4H), 1.45-1.40 (m, 4H), 1.31-1.28 (m, 1H).

General Procedure for Preparation of Compound 4-5′

Reagent MW. amount mol ratio Other Info. Cpd. 4-4 304.17 10.0 g 32.9 mmol 1.0 eq. BrCH₂CO₂tBu 195.05 7.70 g 39.5 mmol 1.2 eq. KOH 56.11 33.0 g  588 mmol  18 eq. Bu₄NHSO₄ 339.53 5.58 g 16.4 mmol 0.50 eq.  toluene 50.0 mL H₂O 50.0 mL Product: (Cpd. 4-5′) 418.31 5.40 g 12.3 mmol Yield: 37%

A mixture of BrCH₂CO₂tBu (7.70 g, 39.5 mmol, 1.2 eq) and Cpd.4-4 (10.0 g, 32.9 mmol, 1.0 eq) in toluene (50.0 mL) was added Bu₄NHSO₄ (5.58 g, 16.4 mmol, 0.50 eq), KOH (33.0 g, 588 mmol, 17.9 eq) in H₂O (50.0 mL) at 0° C., then the mixture was stirred at 25° C. for 16 hrs. TLC (petroleum ether/ethyl acetate=10/1, R_(f)=0.62) show 40% SM remained. H₂O (200 mL) was added, extracted with DCM (200 mL*2), the combined organic layers were washed with brine, dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluted with petroleum ether: EtOAc (40:1) to give Cpd.4-5′ (5.40 g, 37% yield) as yellow oil.

¹H NMR: ET5008-26-P1b1 400 MHz CDCl₃; □7.82 (d, J=7.2 Hz, 1H), 7.30-7.26 (m, 1H), 7.23-7.20 (m, 1H), 6.91-6.88 (m, 1H), 3.97 (s, 2H), 3.53 (t, J=6.8 Hz, 2H), 2.72 (t, J=8.0 Hz, 2H), 1.69-1.59 (m, 4H), 1.58-1.43 (m, 13H).

General Procedure for Preparation of Cpd.4-6

Reagent MW. amount mol ratio Other Info. Cpd. 4-5′ 418.31 5.40 g 12.9 mmol  1.0 eq. Cpd. 2 169.22 2.18 g 12.9 mmol  1.0 eq. CuI 190.45 49.2 mg 258 umol 0.02 eq. PdCl₂(PPh₃)₂ 701.90 181 mg 258 umol 0.02 eq. Et₃N 110 mL Product: (Cpd. 4-6) 459.62 3.00 g 6.20 mmol Yield: 48%

A mixture of Cpd.4-5′ (5.40 g, 12.9 mmol, 1.0 eq) and Cpd.2 (2.18 g, 12.9 mmol, 1.0 eq) in Et₃N (110 mL) was added CuI (49.2 mg, 258 umol, 0.02 eq), PdCl₂(PPh₃)₂ (181 mg, 258 umol, 0.02 eq) at 25° C. under N₂ and stirred at 25° C. for 16 hrs. TLC (petroleum ether/ethyl acetate=1/1, R_(f)=0.3) show that the reaction was complete. Then aq.NH₄Cl (200 mL) was added, extracted with EtOAc (200 mL*3), the combined organic layers were washed with brine, dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluted with petroleum ether: EtOAc (10:1, 1:1) to give Cpd.4-6 (3.00 g, 48% yield) as yellow oil.

¹H NMR: ET5008-32-P1b1 400 MHz CDCl₃; □ 7.41-7.34 (m, 1H), 7.23-7.06 (m, 3H), 4.97-4.87 (m, 1H), 3.97 (s, 2H), 3.53 (t, J=6.8 Hz, 2H), 3.43-3.33 (m, 2H), 2.72 (t, J=8.0 Hz, 2H), 2.64 (J=8.0 Hz, 2H), 1.69-1.59 (m, 4H), 1.55-1.43 (m, 22H).

General Procedure for Preparation of Cpd.4-7

Reagent MW. amount mol ratio Other Info. Cpd. 4-6 459.62 3.00 g 6.53 mmol 1.0 eq. Pd/C 200 mg MeOH 20.0 mL Product: (Cpd. 4-7) 463.65 2.50 g 4.91 mmol Yield: 75%

A mixture of Cpd.4-6 (3.00 g, 6.53 mmol, 1.0 eq) in MeOH (20.0 mL) was added Pd/C (200 mg) and stirred at 25° C. under 50 psi of H₂ for 5 hrs. LC-MS (ET5008-33-P1A4, product: RT=1.04 min) show that the reaction was completed. Then the solution was filtered and concentrated to give Cpd.4-7 (2.50 g, 75% yield) as yellow oil.

¹H NMR: ET5008-33-P1b1 400 MHz CDCl₃; □ 7.13 (s, 4H), 4.54 (s, 1H), 3.96 (s, 2H), 3.52 (t, J=6.8 Hz, 2H), 3.18-3.14 (m, 2H), 2.65-2.57 (m, 4H), 1.75-1.54 (m, 10H), 1.53-1.37 (m, 20H).

General Procedure for Preparation of Cpd.4-10

Reagent MW. amount mol ratio Other Info. Cpd. 4-7 463.65 1.00 g 2.16 mmol 1.0 eq. HCl/EtOAc 30.0 mL 4N Product: (Cpd. 4-10) 343.89 800 mg 2.33 mmol crude

A mixture of Cpd.4-7 (1.00 g, 2.16 mmol, 1.0 eq) in HCl/EtOAc (30.0 mL) at 50° C. and stirred at 50° C. for 0.5 h. LC-MS (ET5008-34-P1A4, product: RT=0.698 min) show that the reaction was completed. The mixture was concentrated to give crude Cpd.4-10 (800 mg) as yellow solid.

General Procedure for Preparation of Comp-05

Reagent MW. amount mol ratio Other Info. Cpd. 4-10 343.89 800 mg 2.33 mmol 1.0 eq. Cpd. R1 131.13 611 mg 4.66 mmol 2.0 eq. Et₃N 101.19 2.36 g 23.3 mmol  10 eq. EtOH 40.0 mL Product: Comp-05 392.49 370 mg 933 umol Yield: 40%

A mixture of Cpd.4-10 (800 mg, 2.33 mmol, 1.0 eq) in EtOH (40.0 mL) was added Et₃N (2.36 g, 23.3 mmol, 10.0 eq) and Cpd.R1 (611 mg, 4.66 mmol, 2.0 eq) at 25° C. Then the solution was stirred at 60° C. for 20 hrs. LC-MS (ET5008-35-P1A1, product: RT=0.81 min) show that the reaction was completed. The solution was concentrated. The residue was purified by prep-HPLC (TFA condition) to give Comp-05 (370 mg, 40% yield) as white solid.

HPLC Separation Method:

Column Luna C18 100*30 5u Condition 0.05% HCl-ACN Begin B 30 End B 60 Gradient Time 12 min 100% B Hold Time  4 min Flow Rate 25 mL/min Injection 12

¹H NMR: ET5008-35-P1b1 400 MHz CDCl₃; 10.46 (br, 1H), 8.35 (s, 1H), 7.74 (s, 1H), 7.12 (s, 4H), 4.12 (s, 2H), 3.59 (t, J=6.0 Hz, 2H 2H), 3.35 (q, J=7.2 Hz, 2H), 2.92 (d, J=5.2 Hz, 3H), 2.65-2.57 (m, 4H), 1.68-1.44 (m, 14H).

For the synthesis of Comp-14 to Comp-34 compounds, general building blocks have been synthezised beforehand:

Building Block 1 (BB-1) N′-[(5Z)-6-iodohex-5-en-1-yl]-Nmethylethanediamide

Step 1:

PPh3 (140 g) and phthalimide (82.5 g) were suspended in dry THF (500.0 mL) and cooled to 0° C. A solution of 5-hexyn-1-ol (50.0 g) and diisopropyl azodicarboxylate (110 mL) in dry THF (100 mL) was then added dropwise over a period of 45 min. The resulting mixture was stirred at 0° C. for 1 h and then at r.t. over night.

THF was removed in vacuo as far as possible. The residue was suspended in PE/EtOAc=9:1 (700 mL) and stirred vigourously. The solvent was decanted off from the precipitated OPPh3. During this process, white needles (product) formed in the decanted solvent, which were filtered off and set aside (F1).

The OPPh3 precipitate was then further washed with PE/EtOAc=9:1 several times. All filtrates were then combined and evaporated in vacuo (F2). The needles from F1 were dissolved in EtOAc (200 mL) and washed with 1N NaOH (2×75 mL) and brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The residue was filtered through a patch of SiO 2 (eluent CH2Cl2). The solvent was removed in vacuo and the oily residue was left standing in the fridge over weekend, after which white needles had been formed. The mixture was diluted with PE, the product was then filtered off, washed with PE and dried in vacuo to afford F1 as white needles. The mother liquor was combined with F2.

The yellow oil of F2 was dissolved in EtOAc (400 mL) and washed with 1 N NaOH (3×150 mL) and brine (50 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO 2 (eluent CH2Cl2). The product containing fractions were combined and evaporated. PE was added to the yellow oily residue, after which a precipitate formed. The mixture was cooled to 0° C., the solid was then filtered off and washed with PE to afford F2 as white solid. The mother liquor was evaporated. PE was added to the oily residue after which a precipitate formed. The mixture was left standing in the fridge for 2 h, the precipitate was then filtered off, washed with PE and dried in vacuo to afford F3 as pale yellow solid.

Step 2:

2-(hex-5-yn-1-yl)-2,3-dihydro-1H-isoindole-1,3-dione (46.3 g), AgNO 3 (8.65 g) and NIS (68.8 g) were placed in a 1 L flask. Dry THF (500 mL) was added, the flask was flushed with argon and wrapped with aluminium foil to protect the reaction from light. The mixture was then stirred under an Ar-atmosphere at r.t. for 16 h. Control by LC/MS showed product.

The reaction mixture was decanted from the formed precipitate, diluted with water (400 mL) and extracted with EtOAc (3×200 mL). The combined organic layers were washed with water (100 mL), sat. Na 2SO3 (3×100 mL) and brine (100 mL), dried over Na2SO4 and concentrated in vacuo. The residue was recrystallized from EtOH to afford F1 as white solid. The mother liquor was evaporated and again recrystallized from EtOH to afford F2 as yellow solid.

Step 3:

2-Methyl-2-butene (29.4 mL) was added dropwise to a 0° C. cold solution of BH 3*SMe2 (2.00 M in THF, 64.4 mL) and stirred at 0° C. for 1 h and then at r.t. for 1 h. The mixture was then added dropwise to a 0° C. cold suspension of 2-(6-iodohex-5-yn-1-yl)-2,3-dihydro-1H-isoindole-1,3-dione (17.5 g) in THF (200 mL). After addition, the resulting mixture was stirred at r.t. for 1 h. Control by LC/MS showed complete consumption of starting material. The reaction mixture was cooled to 0° C., then HOAc (30.0 mL) was added dropwise, stirred for 30 min at 0° C. and then at r.t. over night. Control by LC/MS showed product.

THF was removed in vacuo as far as possible. The residue was then slowly poured into a solution of NaOH (15.0 g) in H₂O (200 mL) and extracted with CH2Cl2 (3×100 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was used for further transformation as such.

Step 4:

2-[(5Z)-6-iodohex-5-en-1-yl]-2,3-dihydro-1H-isoindole-1,3-dione (17.6 g, crude IK-0353/4) was dissolved in MeOH (150 mL). Hydrazine hydrate (6.00 mL) was added and the resulting mixture was stirred at r.t. for 16 h. Control by LC/MS showed product.

MeOH was removed in vacuo. The residue was suspended in CH2Cl2 (300 mL). The solid was filtered off and washed with CH2Cl2 (2×100 mL). The combined filtrates were then washed with water (2×100 mL), dried over Na 2SO4 and concentrated in vacuo to afford the crude product as orange oil which was used for further transformation as such.

Step 5:

Ethylchlorformylformiat (10.0 g) was dissolved in THF (50 mL) and cooled to 0° C. Pyridine (7.70 mL) was added dropwise and the mixture stirred at 0° C. for 30 min. Methylamine (2.0 M in THF, 47.6 mL) was then added dropwise. Stirring was continued at 0° C. for 3 h. Control by TLC (PE/EtOAc=1:3) showed product.

The precipitated salt was filtered off and the filtrate evaporated. The residue was taken up in EtOAc (200 mL), washed with 1N HCl (2×50 mL), dried over Na 2SO4 and concentrated in vacuo to give the product in sufficient purity as brown oil.

Step 6:

(5Z)-6-iodohex-5-en-1-amine (11.15 g) was dissolved in EtOH (200 mL). ethyl (methylcarbamoyl)formate (6.50 g) and NEt3 (8.26 mL) were added and the resulting mixture was stirred at 50° C. for 24 h. Control by LC/MS showed incomplete conversion. Additional (methylcarbamoyl)formate (1.00 g) and NEt3 (4.00 mL) were added and stirring was continued at 50° C. for 24 h. Control by LC/MS showed product.

EtOH was removed in vacuo. The residue was purified by column chromatography on SiO2 (CH2Cl2→CH2Cl2/MeOH=50:1→CH2Cl2/MeOH=20:1). The product containing fractions were combined and evaporated. EtOAc (30 mL) was added to the partly solid residue, treated with sonication and left standing in the fridge over weekend. The precipitate was then filtered off, washed with little ice cold EtOAc and dried in vacuo.

Yield: 10.3 g (67%) pale yellow solid.

Building Block 2 (BB-2) N′-[4-(2-iodophenyl)butyl]-Nmethylethanediamide

Step 1:

PPh3 (95.5 g), phthalimide (56.1 g) and 3-Buten-1-ol (25.0 g) were suspended in dry THF (250 mL) and cooled to 0° C. Diisopropyl azodicarboxylate (75.1 mL) was then added dropwise over a period of 20 min. The resulting mixture was stirred at 0° C. for 30 min and then at r.t. over night. Control by LC/MS showed product.

THF was removed in vacuo as far as possible. The oily residue was diluted with PE/EtOAc=9:1 (400 mL) and stirred vigourously until a precipitate occurred. The precipitated OPPh3 was filtered off and washed extensively with PE/EtOAc=9:1. The combined filtrates were filtered through a patch of SiO 2 and then evaporated. The residue was diluted with PE (200 mL), mixed vigourously and placed in an icebath. The precipitated product was then filtered off and washed with PE to afford the product in sufficient purity as pale yellow solid.

Step 2:

2-(but-3-en-1-yl)-2,3-dihydro-1H-isoindole-1,3-dione (22.1 g) was placed in a 1 L flask under and Ar-atmosphere. 9-BBN (0.5 M in THF, 273 mL) was then added dropwise at 0° C. and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. over night. A solution of Na2CO3 (48.4 g) in water (250 mL) was then added and stirring was continued at r.t. for 30 min. Then 2-Iodo-phenylamine (20.0 g) and PdCl2(PPh3)2 (2.80 g) was added and the mixture heated to 50° C. for 4 h. Control by LC/MS showed product.

The reaction mixture was diluted with EtOAc (200 mL) and the layers separated. The aqueous layer was extracted with EtOAc (300 mL) and the combined organic layers were washed with brine (200 mL) and dried over Na2SO4. The residue was purified by column chromatography on SiO2 (PE/EtOAc=6:4).

Step 3:

2-[4-(2-aminophenyl)butyl]-2,3-dihydro-1H-isoindole-1,3-dione (22.0) was dissolved in acteone (100 mL). Then water (200 mL) and conc. H2SO4 (13.9 mL) were added and the resulting suspension was cooled to 0° C. A solution of NaNO2 (5.23 g) in water (50 mL) was added dropwise and the mixture stirred at 0° C. for 30 min. Then a solution of KI (37.2 g) in water (50 mL) was added dropwise, the reaction mixture warmed to r.t. and stirred for 20 h. Control by LC/MS showed product.

The reaction mixture was diluted with sat. Na 2503 (200 mL) and extracted with EtOAc (3×200 mL). The combined organic layers were washed with brine (150 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=8:2).

Step 4:

2-[4-(2-iodophenyl)butyl]-2,3-dihydro-1H-isoindole-1,3-dione (21.2 g) was suspended in MeOH (300 mL). Hydrazine hydrate (5.10 mL) was added and the resulting mixture was stirred at r.t. for 3d. Control by LC/MS showed product.

MeOH was removed in vacuo. The residue was suspended in CH2Cl2 (200 mL). The solid was filtered off and washed with CH2Cl2 (100 mL). The combined filtrates were then washed with water (2×100 mL). The combined aqueous layers were reextracted with CH2Cl2 (50 mL) and the combined organic layers were then dried over Na2SO4 and concentrated in vacuo to afford the product in sufficient purity as yellow oil.

Step 5:

Ethylchlorformylformiat (10.0 g) was dissolved in THF (50 mL) and cooled to 0° C. Pyridine (7.70 mL) was added dropwise and the mixture stirred at 0° C. for 30 min. Methylamine (2.0 M in THF, 47.6 mL) was then added dropwise. Stirring was continued at 0° C. for 3 h. Control by TLC (PE/EtOAc=1:3) showed product.

The precipitated salt was filtered off and the filtrate evaporated. The residue was taken up in EtOAc (200 mL), washed with 1N HCl (2×50 mL), dried over Na 2SO4 and concentrated in vacuo to give the product in sufficient purity as brown oil.

Step 6:

4-(2-iodophenyl)butan-1-amine (11.0 g, crude IK-0355710) was dissolved in EtOH (100 mL). Ethyl (methylcarbamoyl) formate (5.76 g) and NEt3 (6.67 mL) were added and the resulting mixture was stirred at 50° C. for 18 h. Control by LC/MS showed product.

The reaction mixture was cooled to r.t. and EtOH was removed in vacuo. The residue was filtered through a patch of SiO2 (CH2Cl2/MeOH=98:2). Further purification by recrystallization from EtOAc.

Yield: 7.76 g (54%) beige solid.

Building Block 4 (BB-4) 2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid

Step 1

NaH (60% in mineral oil, 771 mg) was suspended in dry THF (20.0 mL). The mixture was cooled to 0° C., then 6-Hepten-1-ol (1.18 mL) was added. Stirring was continued at 0° C. for 30 min, then a solution of bromoacetic acid (1.34 g) in THF (10.0 mL) was added dropwise. After complete addition, the ice bath was removed and stirred for 15 min, then the mixture was heated to 70° C. for 1.5 h. Control by TLC (PE/EtOAc=1:1) showed product.

The reaction mixture was poured into 1N NaOH (50 mL) and extracted with EtOAc (2×30 mL). The combined organic layers contained no product and were discarded. The aqueous layer was carefully acidified with conc. HCl and then again extracted with EtOAc (3×30 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to afford the product in sufficient purity as colorless oil.

Step 2

1,1′-Carbonyldiimidazole (15.6 g) was suspended in THF (200 mL). A solution of 2-(hept-6-en-1-yloxy)acetic acid (15.1 g) in THF (20 mL) was then added dropwise and the resulting mixture was stirred at r.t. for 6 h. THF was then removed in vacuo and MeOH (200 mL) was added to the residue. The mixture was stirred at r.t. for 3d. Control by TLC (PE/EtOAc=9:1) showed product.

MeOH was removed in vacuo. PE (200 mL) was added to the residue and stirred vigourously for 5 min. The solvent was then decanted off from a thick, oily residue, which was further washed with PE (2×100 mL) and then discarded. The combined PE fractions were washed with 1N HCl (100 mL) and 1N NaOH (100 mL), dried over Na2SO4 and concentrated in vacuo to afford the product in sufficient purity as colorless liquid.

Step 3

Methyl 2-(hept-6-en-1-yloxy)acetate (2.88 g) was placed in 100 mL flask and cooled to 0° C. under and Aratmosphere. 9-BBN (0.5 M in THF, 38.7 mL) was then added dropwise and the resulting mixture stirred at 0° C. for 30 min and then at r.t. for 2 h. A solution of Na 2CO3 (6.84 g) in water (30.0 mL) was then added and stirring was continued at r.t. for 30 min. Then N′-[(5Z)-6-iodohex-5-en-1-yl]-N-methylethanediamide (BB-1, 4.00 g) and PdCl2(PPh3)2 (453 mg) were added and the mixture heated to 50° C. for 1.5 h. Control by LC/MS showed product.

The reaction mixture was cooled to r.t. and the layers were separated. The aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc 1:1).

Step 4

Methyl 2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetate (400 mg) was suspended in MeOH (20.0 mL). NaOH (3N, 5.00 mL) was added and the resulting mixture was stirred at r.t. for 15 min. Control by LC/MS showed product.

The reaction mixture was poured into 1N HCl (30 mL). The precipitated product was filtered off, washed with water and dried in vacuo.

Yield: 869 mg (86%) beige solid.

Building Block 6 (BB-6) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid

Step 1:

NaH (60% in mineral oil, 15.2 g) was suspended in dry THF (250 mL). The mixture was cooled to 0° C., then allylalcohol (11.8 mL) was added. Stirring was continued at 0° C. for 30 min, then a solution of bromoacetic acid (26.3 g) in THF (50.0 mL) was added dropwise. After complete addition, the ice bath was removed and stirred for 15 min, the mixture was then heated to 70° C. for 3 h and stirred at r.t. over night.

The reaction mixture was poured into water (250 mL) and extracted with EtOAc (2×100 mL). The combined organic layers contained no product and were discarded. The aqueous layer was carefully acidified with conc. HCl and then again extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to afford the product in sufficient purity as pale brown liquid.

Step 2:

1,1′-Carbonyldiimidazole (30.7 g) was suspended in THF (200 mL). 2-(prop-2-en-1-yloxy)acetic acid (crude IK-0352/9) was then added dropwise and the resulting mixture was stirred at r.t. for 7 h. THF was then removed in vacuo and MeOH (200 mL) was added to the residue. The mixture was stirred at r.t. over night. Control by TLC (PE/EtOAc=8:2) showed product.

MeOH was removed in vacuo. PE (200 mL) was added to the residue and stirred vigourously for 5 min. The solvent was then decanted off from a thick, oily residue, which was further washed with PE (2×100 mL). Control by TLC showed most of the product remaining in the oily residue, which was thus washed with MTBE (4×100 mL). The PE and MTBE layers were combined and washed with 1N HCl (3×100 mL) and brine (50 mL), dried over Na2SO4 and concentrated in vacuo to afford the product in sufficient purity as pale yellow liquid.

Step 3:

methyl 2-(prop-2-en-1-yloxy)acetate (1.30 g) was placed in a 100 mL flask and cooled to 0° C. under and Aratmosphere. 9-BBN (0.5 M in THF, 25.0 mL) was then added dropwise and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 2 h. A solution of Na 2CO3 (4.41 g) in water (25.0 mL) was then added and stirring was continued at r.t. for 30 min. Then N′-[4-(2-iodophenyl)butyl]-N-methylethanediamide (BB-2, 3.00 g) and PdCl 2(PPh3)2 (292 mg) were added and the mixture heated to 50° C. for 4 h and then stirred at r.t. overnight. Control by LC/MS showed incomplete conversion. Additional methyl 2-(prop-2-en-1-yloxy)acetate (650 mg) was placed in a separate flask under an Ar-atmosphere. 9-BBN (0.5 M in THF, 12.5 mL) was added at r.t. and the mixture stirred at r.t. for 2 h. A sat. solution of Na 2CO3 (10 mL) was added and stirring was continued at r.t. for 30 min. The mixture was then added to the above reaction mixture. After adding fresh PdCl2(PPh3)2 (200 mg), the mixture was stirred at 50° C. for 2 h. Control by LC/MS showed product.

The reaction mixture was cooled to r.t. and the layers were separated. The aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc 3:7).

Step 4:

methyl 2-[3-(2-{4-[(methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetate (2.04 g) was dissolved in THF (30 mL). NaOH (3N, 30 mL) and MeOH (20 mL) were added and the resulting mixture was stirred at r.t. for 5 min. Control by LC/MS showed product.

The reaction mixture was acidified with 6N HCl and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by a short column on SiO2 (CH2Cl2/MeOH=9:1).

Yield: 1.56 g (80%) beige solid.

Building Block 8 (BB-8) N′-[(5Z)-13-hydroxytridec-5-en-1-yl]-Nmethylethanediamide

Step 1:

6-Hepten-1-ol (3.00 g) and imidazole (3.57 g) were dissolved in DMF (20.0 mL). TIPSCl (6.18 mL) was added and the resulting mixture was stirred at 60° C. for 6 h. Control by TLC (PE/EtOAc=8:2) showed almost complete conversion.

The reaction mixture was diluted with water (100 mL) and extracted with MTBE (3×40 mL). The combined organic layers were washed with 1N HCl (2×50 mL) and brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=95:5).

Step 2:

(hept-6-en-1-yloxy)tris(propan-2-yl)silane (1.57 g) was placed in 100 mL flask and cooled to 0° C. under and Aratmosphere. 9-BBN (0.5 M in THF, 14.5 mL) was then added dropwise and the resulting mixture stirred at 0° C. for 30 min and then at r.t. for 2 h. A solution of Na 2CO3 (2.56 g) in water (15.00 mL) was then added and stirring was continued at r.t. for 30 min. then N′-[(5Z)-6-iodohex-5-en-1-yl]-N-methylethanediamide (BB-1, 1.50 g) and PdCl2(PPh3)2 (170 mg) were added and the mixture heated to 50° C. for 2 h. Control by LC/MS showed product.

The reaction mixture was cooled to r.t. and the layers were separated. The aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine (30 mL), dried over Na 2SO4 and concentrated in vacuo. The residue was filtered through a patch of SiO 2 (PE/EtOAc=4:6). The so obtained crude product was used for further transformation as such.

Step 3:

(hept-6-en-1-yloxy)tris(propan-2-yl)silane (2.20 g, crude 1K-0357/16) was dissolved in THF (50 mL) and cooled to 0° C. TBAF*3H₂O (2.29 g) was added and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 6 h. Control by TLC (PE/EtOAc=1:1) and LC/MS showed complete conversion.

The reaction mixture was poured into water (100 mL) and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was passed through a short column on SiO2 (PE/EtOAc=1:1→EtOAc).

Yield: 1.11 g (77%) beige solid.

Building Block 9 (BB-9) N′-{4-[2-(3-hydroxypropyl)phenyl]butyl}-Nmethylethanediamide

Step 1:

2-Propen-1-ol (3.00 g) and imidazole (7.03 g) were dissolved in DMF (20.0 mL). TIPSCl (14.4 mL) was added and the resulting mixture was stirred at 60° C. for 6 h. Control by TLC (PE/EtOAc=8:2) showed almost complete conversion. The reaction mixture was diluted with water (100 mL) and extracted with MTBE (3×40 mL). The combined organic layers were washed with 1N HCl (2×50 mL) and brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=95:5).

Step 2:

(prop-2-en-1-yloxy)tris(propan-2-yl)silane (1.33 g) was placed in a 100 mL flask and cooled to 0° C. under and Ar-atmosphere. 9-BBN (0.5 M in THF, 14.2 mL) was then added dropwise and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 2 h.

A solution of Na 2CO3 (2.21 g) in water (15.0 mL) was then added and stirring was continued at r.t. for 30 min. Then N′-[4-(2-iodophenyl)butyl]-N-methylethanediamide (1.50 g) and PdCl 2(PPh3)2 (146 mg) were added and the mixture heated to 50° C. for 3 h. Control by LC/MS showed product. The reaction mixture was cooled to r.t. and the layers were separated. The aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The residue was passed through a short column of SiO 2 (PE/EtOAc 1:1). The still crude product was then used for further transformation as such.

Step 3:

N-methyl-N′-{4-[2-(3-{[tris(propan-2-yl)silyl]oxy}propyl)phenyl]butyl}ethanediamide (1.87 g, crude IK-0357/17) was dissolved in THF (50 mL) and cooled to 0° C. TBAF*3H₂O (1.97 g) was added and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 16 h. Control by LC/MS showed complete conversion. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was passed through a short column on SiO2 (PE/EtOAc=1:1→EtOAc).

Yield: 911 mg (75%) beige solid.

Building Block 11 (BB-11) N′-[(5Z)-13-(2-aminoethoxy)tridec-5-en-1-yl]-Nmethylethanediamide

Step 1:

NaH (60% in mineral oil, 7.71 g) was suspended in dry THF (200 mL). The mixture was cooled to 0° C., then 6-Hepten-1-ol (11.8 mL) was added. Stirring was continued at 0° C. for 30 min, then a solution of bromoacetic acid (13.4 g) in THF (100 mL) was added dropwise. After complete addition, the ice bath was removed and stirred for 15 min, then the mixture was heated to 70° C. for 3 h. Control by TLC (PE/EtOAc=1:1) showed product.

The reaction mixture was poured into 1N NaOH (300 mL) and extracted with EtOAc (2×100 mL). The combined organic layers contained no product and were discarded. The aqueous layer was carefully acidified with conc. HCl and then again extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to afford the product in sufficient purity as pale brown oil.

Step 2:

1,1′-Carbonyldiimidazole (15.6 g) was suspended in THF (200 mL). A solution of 2-(hept-6-en-1-yloxy)acetic acid (15.1 g) in THF (20 mL) was then added dropwise and the resulting mixture was stirred at r.t. for 6 h. THF was then removed in vacuo and MeOH (200 mL) was added to the residue. The mixture was stirred at r.t. for 3d. Control by TLC (PE/EtOAc=9:1) showed product.

MeOH was removed in vacuo. PE (200 mL) was added to the residue and stirred vigourously for 5 min. The solvent was then decanted off from a thick, oily residue, which was further washed with PE (2×100 mL) and then discarded. The combined PE fractions were washed with 1N HCl (100 mL) and 1N NaOH (100 mL), dried over Na2SO4 and concentrated in vacuo to afford the product in sufficient purity as colorless liquid.

Step 3:

methyl 2-(hept-6-en-1-yloxy)acetate (5.00 g) was dissolved in CH 2Cl2 (100 mL) and cooled to 0° C. DIBALH (1.00 M in CH2Cl2, 61.7 mL) was added dropwise, and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. over night. Control by TLC (PE/EtOAc=8:2) showed complete conversion.

The reaction mixture was cooled to 0° C. and carefully quenched with sat. aqueous Na 2SO4. The mixture was then diluted with CH2Cl2 (100 mL), stirred vigourously for 20 min and then filtered through celite. The filtercake was washed with CH2Cl2 several times. The combined filtrates were concentrated in vacuo to afford the product in sufficient purity as colorless liquid.

Step 4:

PPh3 (7.17 g), phthalimide (4.21 g) and 2-(hept-6-en-1-yloxy)ethan-1-ol (4.12 g) were suspended in dry THF (100 mL) and cooled to 0° C. Diisopropyl azodicarboxylate (5.79 mL) was then added dropwise over a period of 20 min. The resulting mixture was stirred at 0° C. for 30 min and then at r.t. over night.

THF was removed in vacuo as far as possible. The oily residue was diluted with PE/EtOAc=9:1 (200 mL) and stirred vigourously until a precipitate occurred. The precipitated OPPh3 was filtered off and washed extensively with PE/EtOAc=9:1. The combined filtrates were filtered through a patch of SiO2 (eluent PE/EtOAc=9:1) and evaporated. The residue was purified by column chromatography on SiO2 (PE/EtOAc=8:2).

Step 5:

2-[2-(hept-6-en-1-yloxy)ethyl]-2,3-dihydro-1H-isoindole-1,3-dione (2.22 g) was placed in 100 mL flask and cooled to 0° C. under and Ar-atmosphere. 9-BBN (0.5 M in THF, 19.3 mL) was then added dropwise and the resulting mixture stirred at 0° C. for 30 min and then at r.t. for 2 h. A solution of Na2CO3 (3.42 g) in water (20.0 mL) was then added and stirring was continued at r.t. for 30 min. then N′-[(5Z)-6-iodohex-5-en-1-yl]-N-methylethanediamide (BB-1, 2.00 g) and PdCl 2(PPh3)2 (226 mg) were added and the mixture heated to 50° C. for 1.5 h. Control by LC/MS showed product.

The reaction mixture was cooled to r.t. and the layers were separated. The aqueous layer was extracted with EtOAc (2×30 mL). The combined organic layers were washed with brine (30 mL), dried over Na 2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=4:6).

Step 6:

N′-[(5Z)-13-[2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)ethoxy]tridec-5-en-1-yl]-N-methylethanediamide (2.36 g) was suspended in MeOH (100 mL). Hydrazine hydrate (486 μL) was added and the resulting mixture was stirred at r.t. for 16 h. Control by LC/MS showed product.

MeOH was removed in vacuo. The residue was suspended in CH 2Cl2/7N NH3 in MeOH=9:1 (100 mL) and filtered through as patch of SiO2 and further eluated with CH2Cl2/7N NH3 in MeOH=9:1 (300 mL). The filtrate was concentrated in vacuo to afford 1.68 g of the crude product. 60 mg were subjected to purification by preparative TLC (CH2Cl2/7N NH3 in MeOH=9:1). The rest of the crude material was used for further transformations as such.

Yield: 41 mg (2%) pale yellow solid (purified).

Compound 14 (Comp-14) N-methyl-N′-[(5Z)-13-[(1H-1,2,3,4-tetrazol-5-yl)methoxy]tridec-5-en-1-yl] ethanediamide

Step 1

NaH (60% in mineral oil, 7.71 g) was suspended in dry THF (200 mL). The mixture was cooled to 0° C., then 6-Hepten-1-ol (11.8 mL) was added. Stirring was continued at 0° C. for 30 min, then a solution of bromoacetic acid (13.4 g) in THF (100 mL) was added dropwise. After complete addition, the ice bath was removed and stirred for 15 min, then the mixture was heated to 70° C. for 3 h. Control by TLC (PE/EtOAc=1:1) showed product. The reaction mixture was poured into 1N NaOH (300 mL) and extracted with EtOAc (2×100 mL). The combined organic layers contained no product and were discarded. The aqueous layer was carefully acidified with conc. HCl and then again extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to afford the product in sufficient purity as pale brown oil.

Yield: 14.1 g (93%) pale brown oil

Step 2

A mixture of 2-(hept-6-en-1-yloxy)acetic acid (3.00 g) and SOCl 2 (15.00 mL) was heated to 70° C. for 1 h. Excess SOCl2 was then removed in vacuo and the residue was taken up in dichloroethane (15.0 ml). Then ammonia was slowly bubbled through the solution for 5 min. The reaction mixture diluted with water (50 mL) and extracted with CH2Cl2 (3×30 mL). The combined organic layers were washed with sat. NaHCO₃(30 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo to afford the product in sufficient purity as white solid. m=2,16 g (y=62%). Analog in TLC to IK-0367/1

Step 3

2-(hept-6-en-1-yloxy)acetamide (2,61 g) was dissolved in CH2Cl2 (50 mL). NEt3 (6.35 mL) was added and the mixture was cooled to 0° C. A solution of POCl3 (1.54 mL) in CH2Cl2 (4 mL) was slowly added. Stirring was then continued at 0° C. for 15 min. Control by TLC (PE/EtOAc=8:2) showed product.

sat. NaHCO₃(5.00 mL) was added at 0° C. and stirred for 30 min at that temperature. The mixture was allowed to come to r.t., diluted with water (15.0 mL) and extracted with CH2Cl2 (3×20 mL). The combined organic layers were washed with sat. NaHCO₃(10.0 mL) and brine (10.0 mL), dried over Na2SO4 and then filtered through a pad of SiO2 (eluent CH2Cl2). The product was obtained after evaporation in sufficient purity as colorless oil. m=2.08 g, y=89%.

Step 4

2-(hept-6-en-1-yloxy)acetonitrile was placed in a 10 mL flask and cooled to 0° C. under and Ar-atmosphere. 9-BBN (0.5 M in THF, 1.63 mL) was then added dropwise and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 2 h. A solution of Na2CO3 (288 mg) in degazed water (1 mL) was then added and stirring was continued at r.t. for 30 min. Then N′-[(5Z)-6-iodohex-5-en-1-yl]-N-methylethanediamide (168 mg) and PdCl2(PPh3)2 (19 mg) were added and the mixture heated to 50° C. overnight. Water was added and mixture was extracted with DCM. Organic layer was dried over MgSO4, filtered and solvent evaporated. Mixture was purified by preparative TLC (DCM/MeOH 95/5). m=70 mg, y=38%.

Step 5

N′-[(5Z)-13-(cyanomethoxy)tridec-5-en-1-yl]-N-methylethanediamide, natrium azide and triethylamine hydrochloride were dissolved in THF and the reaction mixture stirred at 70° C. overnight.

Water and ethyl acetate were added. The mixture was acidified with HCl3N. The aqueous layer (acid pH) is then extracted with ethyl acetate (×3), and the combined organic layer washed with brine. The organic layer was dried over MgSO4, filtered and solvent removed under vacuo. m=82 mg. Product was purified by preparative TLC (DCM/MeOH 95/5)

Yield: 8 mg (10%), as white powder.

Compound 15 (Comp-15) N′-(4-{2-[3-(carbamoylmethoxy)propyl]phenyl}butyl)-N-methylethanediamide

250 mg (0.72 ol) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid (BB-6) and 164.1 mg (0.86 mmol) EDCI were dissolved in 20 ml DCM. 36.5 mg (2.14 mmol,

5.35 ml) ammonia (0.4 M in THF) were added and the mixture was stirred at rt over the weekend.

The mixture was poured into 50 ml water and extracted with DCM (3×50 ml). The combined organic layers were dried over Na2SO4 and concentrated.

Yield: 50 mg (20%), white solid.

Compound 16 (Comp-16) N-Methyl-N′-[(5Z)-13-[(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy]tridec-5-en-1-yl]ethanediamide

Step 1:

NaH (60% in mineral oil, 7.71 g) was suspended in dry THF (200 mL). The mixture was cooled to 0° C., then 6-Hepten-1-ol (11.8 mL) was added. Stirring was continued at 0° C. for 30 min, then a solution of bromoacetic acid (13.4 g) in THF (100 mL) was added dropwise. After complete addition, the ice bath was removed and stirred for 15 min, then the mixture was heated to 70° C. for 3 h. Control by TLC (PE/EtOAc=1:1) showed product. The reaction mixture was poured into 1N NaOH (300 mL) and extracted with EtOAc (2×100 mL). The combined organic layers contained no product and were discarded. The aqueous layer was carefully acidified with conc. HCl and then again extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to afford the product in sufficient purity as pale brown oil.

Step 2:

1,1′-Carbonyldiimidazole (15.6 g) was suspended in THF (200 mL). A solution of 2-(hept-6-en-1-yloxy)acetic acid (15.1 g) in THF (20 mL) was then added dropwise and the resulting mixture was stirred at r.t. for 6 h. THF was then removed in vacuo and MeOH (200 mL) was added to the residue. The mixture was stirred at r.t. for 3d. Control by TLC (PE/EtOAc=9:1) showed product. MeOH was removed in vacuo. PE (200 mL) was added to the residue and stirred vigourously for 5 min. The solvent was then decanted off from a thick, oily residue, which was further washed with PE (2×100 mL) and then discarded. The combined PE fractions were washed with 1N HCl (100 mL) and 1N NaOH (100 mL), dried over Na2SO4 and concentrated in vacuo to afford the product in sufficient purity as colorless liquid.

Step 3:

500 mg (2.68 mmol) and 1.34 g (26.9 mmol, 1.30 ml) hydrazine hydrate were dissolved in 5 ml EtOH and stirred at 70° C. for 4.5 h (→clear solution).

-   -   LC/MS: GH-0513/1-1     -   TLC (EA/PE 1:1): Complete consumption of starting material

The mixture was evaporated to dryness

Step 4:

500 mg (2.68 mmol) 2-(Hept-6-en-1-yloxy)acetohydrazide (GH-0513/1) were dissolved in 3 ml AcOH. 653.3 mg (8.05 mmol) potassium cyanate dissolved in 3 ml water were added and the mixture was stirred at rt for 1.5 h (a yellow solution). The mixture was evaporated to dryness.

Step 5:

The oily residue was dissolved in 10 ml 2M NaOH and heated to reflux for 2 h.→LC/MS: GH-0515/1-2: Complete consumption of intermediate 1. The mixture was acidified using conc. HCl and extracted with EA (3×20 ml). The combined organic layers were dried over Na2SO4 and concentrated to dryness. The crude solid was recrystallized from ACN.

Step 6:

Under Argon atmosphere 92.0 mg (0.44 mmol) 3-[(Hept-6-en-1-yloxy)methyl]-4,5-dihydro-1H-1,2,4-triazol-5-one (GH-0515/1) dissolved in 2 ml anhydrous THF were added to a solution of 88.5 mg (0.73 mmol, 1.45 ml) 9BBN (0.5M in THF) and the mixture was stirred at rt over night. A solution of 153.8 mg (1.45 mmol) Na2CO3 in 1 ml water were added and stirring at rt was continued for 15 min. Then 90.0 mg (0.29 mmol) N′-[(5Z)-6-iodohex-5-en-1-yl]-methylethanediamide (IK-0356/2) dissolved in 2 ml THF and 10.2 mg (14.5 μmol) PdCl 2(PPh3)2 were added and the mixture was heated to 50° C. for 4 h (à yellow biphasic mixture).

-   -   LC/MS: GH-0516/1-1: Product was detected

The organic layer was separated via pipette and evaporated to dryness. The crude product was purified via flash column chromatography on silica gel (DCM/MeOH 20:1 à 9:1, Rf of possible product: 0.62). Recrystallization from CAN.

Yield: 51 mg (0.13 mmol, 45%).

Compound 17 (Comp-17) N-methyl-N′-[(5Z)-13-[(phenylcarbamoyl)methoxy]tridec-5-en-1-yl]ethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50.0 mg, 140.3 μmol), Aniline (26 μl, 280.5 μmol), HBTU (53.4 mg, 140.3 μmol) and DMAP (1.7 mg, 14.0 μmol) were placed in a G16 vial. DMF (2.00 ml) and NEt 3 (78.0 μl, 561.1 μmol) were added and the resulting mixture was stirred at r.t. for 16 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (20 ml) and was extracted with Et 2O (3×20 ml). The combined organic layers were washed with sat. NaHCO₃(20 ml) and brine (10 ml), dried over Na2SO4 and concentrated in vacuo. The product was lyophilized.

Yield: 52 mg (87%), white solid.

Compound 18 (Comp-18) N-methyl-N′-[(5Z)-13-{[(oxan-4-yl)carbamoyl]methoxy}tridec-5-en-1-yl] ethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50.0 mg, 140.3 μmol), 4-Aminotetrahydropyran (29 μl, 280.5 μmol), HBTU (53.4 mg, 140.3 μmol) and DMAP (1.7 mg, 14.0 μmol) were placed in a G16 vial. DMF (2.00 ml) and NEt3 (78.0 μl, 561.1 μmol) were added and the resulting mixture was stirred at r.t. for 16 h. Control by LC/MS showed product. The reaction mixture was diluted with water (20 ml) and was extracted with Et2O (3×20 ml). The combined organic layers were washed with sat. NaHCO₃(20 ml) and brine (10 ml), dried over Na2SO4 and concentrated in vacuo. The product was lyophilized.

Yield: m=58 mg (94%) white solid.

Compound 19 (Comp-19) N-methyl-N′-[(5Z)-13-{[(1,3-oxazol-2-yl)carbamoyl]methoxy}tridec-5-en-1-yl]ethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50.0 mg, 140.3 μmol), 1,3-Oxazol-2-amine (24 mg, 280.5 μmol), HBTU (53.4 mg, 140.3 μmol) and DMAP (1.7 mg, 14.0 μmol) were placed in a G16 vial. DMF (2.00 ml) and NEt 3 (78.0 μl, 561.1 μmol) were added and the resulting mixture was stirred at r.t. for 16 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (20 ml) and was extracted with Et 2O (3×20 ml). The combined organic layers were washed with sat. NaHCO₃(20 ml) and brine (10 ml), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (DCM/MeOH=95:5).

Yield: 11 mg (19%), white solid.

Compound 20 (Comp-20) N′-[(5Z)-13-{[(4-methoxyphenyl)carbamoyl]methoxy}tridec-5-en-1-yl]-Nmethylethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50.0 mg, 140.3 μmol), p-Anisidine (35 mg, 280.5 μmol), HBTU (53.4 mg, 140.3 μmol) and DMAP (1.7 mg, 14.0 μmol) were placed in a G16 vial. DMF (2.00 ml) and NEt3 (78.0 μl, 561.1 μmol) were added and the resulting mixture was stirred at r.t. for 16 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (20 ml) and was extracted with Et 2O (3×20 ml). The combined organic layers were washed with sat. NaHCO₃ (20 ml) and brine (10 ml), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (DCM/MeOH=98:2).

Yield: 18 mg (28%), beige solid.

Compound 21 (Comp-21) N-Methyl-N′-[4-(2-{3-[(phenylcarbamoyl)methoxy]propyl}phenyl)butyl] ethanediamide

40 mg (0.11 mmol) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid (BB-6), 26.3 mg (0.14 mmol) EDCI and 11.7 mg (0.13 mmol, 11.5 μl) aniline were dis¬sol¬ved in 3 ml DCM and stirred at rt of the weekend (clear solution).

The mixture was evaporated to dryness and purified via pTLC (1 mm, DCM/MeOH 20:1, Rf of possible product: 0.54).

Yield: 24 mg (51%), white solid.

Compound 22 (Comp-22) N-Methyl-N′-[4-(2-{3-[2-oxo-2-(pyrrolidin-1-yl)ethoxy]propyl}phenyl)butyl] ethanediamide

50 mg (0.14 mmol) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid (BB-6), 32.8 mg (0.17 mmol) EDCI and 20.3 mg (0.29 mmol, 23.4 μl) Pyrrolidine were dissolved in 3 ml DCM and stirred at rt for 1.5 h (clear solution).

The mixture was evaporated to dryness and purified via pTLC (1 mm, DCM/MeOH 10:1, Rf of possible product: 0.46).

Yield: 20 mg (35%), white solid.

Compound 23 (Comp-23) N-Methyl-N′-[4-(2-{3-[2-(morpholin-4-yl)-2-oxoethoxy]propyl}phenyl)butyl] ethanediamide

50 mg (0.14 mmol) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid (BB-6, IK-0358/6), 32.8 mg (0.17 mmol) EDCI and 24.9 mg (0.29 mmol, 24.9 μl) morpholine were dissolved in 3 ml DCM and stirred at rt over the weekend (clear solution).

The mixture was evaporated to dryness and purified via pTLC (1 mm, DCM/MeOH 10:1, Rf of possible product: 0.48).

Yield: 34 mg (58%), white solid.

Compound 24 (Comp-24) N′-[(5Z)-13-{[(benzenesulfonyl)carbamoyl]methoxy}tridec-5-en-1-yl]-Nmethylethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50.0 mg) was dissolved in THF (2.00 mL). 1,1′-Carbonyldiimidazole (29.6 mg) was added and the resulting mixture was stirred at r.t. for 2 h. Then DBU (52.9 μL) and benzenesulfonamide (33.1 mg) were added and stirring was continued at r.t. for 25 h. Control by LC/MS showed OMT-121.

The reaction mixture was diluted with water (30 mL) and extracted with CH 2Cl2 (3×20 mL). The combined organic layers were washed with 1N HCl (20 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 44 mg (63%), white solid.

Compound 25 (Comp-25) 4-{2-[3-(2-{4-[(methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetamido}benzoic acid

Step 1:

50 mg (0.14 mmol) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid (BB-6, IK-0358/6), 32.8 mg (0.17 mmol) EDCI and 43.1 mg (0.29 mmol) Methyl 4-aminobenzoate were dissolved in 3 ml DCM and stirred at rt for 1.5 h (clear solution).

The mixture was evaporated to dryness and purified via pTLC (1 mm, EA/PE 4:1, R f of possible product: 0.31)

Step 2:

To a solution of 48 mg (0.10 mmol) N-Methyl-N′-{4-[2-(3-{[(pyridin-2-yl)carbamoyl]methoxy}propyl)phenyl]butyl}ethanediamide (GH-0498/1) in 2 ml THF 16.7 mg (0.40 mmol) LiOH monohydrate dissolved in 0.5 ml water were added and the mixture was stirred at rt over night (a biphasic mixture).

The mixture was poured into 1 N HCl solution (10 ml) and extracted with DCM (3×20 ml). The combined organic layers were dried over Na2SO4 and concentrated to dryness. The crude product was purified via pTLC (DCM/MeOH/FA 100:10:1, Rf of possible product: 0.43).

Yield: 10 mg (21%), white solid.

Compound 26 (Comp-26) N′-[(5Z)-13-[2-(4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)-2-oxoethoxy]tridec-5-en-1-yl]-Nmethylethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50 mg), DCC (34.7 mg), DMAP (22.3 mg) and 2,4(3H,5H)-Furandione (15.4 mg) were placed in a G16 vial. CH2Cl2 (3.00 mL) was added and the resulting mixture was stirred at r.t. for 18 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (30 mL) and extracted with CH 2Cl2 (3×20 mL). The combined organic layers were washed with brine (10 mL), dried over Na 2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=9:1).

Yield: 20 mg (33%), beige solid.

Compound 27 (Comp-27) N′-[4-(2-(3-[2-(hydroxymethyl) phenoxyl]propyl)phenyl)butyl]-N-methylethanediamide

Step 1:

Salicylaldehyde (2.00 g) and imidazole (2.79 g) were dissolved in DMF (20.0 mL). TIPSCl (5.96 mL) was added and the resulting mixture was stirred at 60° C. for 2d. Control by TLC (PE/EtOAc=95:5) and LC/MS showed incomplete conversion. Additional TIPSCl (2.00 mL) was added and stirring was continued at 60° C. for 3d. Control by TLC (PE/EtOAc=95:5) and LC/MS showed almost complete conversion. The reaction mixture was diluted with water (100 mL) and extracted with MTBE (3×40 mL). The combined organic layers were washed with 1N NaOH (30 mL) and brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=95:5).

Yield: 3.54 g (78%) pale yellow liquid

Step 2:

2-{[tris(propan-2-yl)silyl]oxy}benzaldehyde (3.54 g) was dissolved in EtOH (30.0 mL) and cooled to 0° C. NaBH4 (481 mg) was added and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 18 h. Control by TLC (PE/EtOAc=8:2) and LC/MS showed product. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=8:2).

Yield: 2.73 g (77%) yellow oil

Step 3:

(2-{[tris(propan-2-yl)silyl]oxy}phenyl)methanol (400 mg) was dissolved in dry THF (15 mL). NaH (60% in mineral oil, 85.6 mg) was added and the mixture stirred at r.t. for 15 min. Allylbromide (309 μL) was then added and the resulting mixture was stirred at r.t. over night. Control by LC/MS showed product. The reaction mixture was diluted with water (50 mL) and extracted with CH2Cl2 (3×30 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The so obtained crude product was used for further transformation as such.

Yield: 480 mg (crude) yellow oil

Step 4:

{2-[(prop-2-en-1-yloxy)methyl]phenoxy}tris(propan-2-yl)silane (178 mg) was placed in a 10 mL flask and cooled to 0° C. under and Ar-atmosphere. 9-BBN (0.5 M in THF, 1.38 mL) was then added dropwise and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 2 h. A solution of Na2CO3 (147 mg) in water (1.50 mL) was then added and stirring was continued at r.t. for 30 min. Then N′-[4-(2-iodophenyl)butyl]-N-methylethanediamide (BB-2, 100 mg) and PdCl2(PPh3)2 (9.7 mg) were added and the mixture heated to 50° C. for 3 h. Control by LC/MS showed product. The reaction mixture was cooled to r.t., diluted with water (20 mL) and the layers were separated. The aqueous layer was extracted with EtOAc (15 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was passed through a short column of SiO2 (PE/EtOAc 1:1). The still crude product was then used for further transformation as such.

Yield: 235 mg (crude) yellow oil.

Step 5:

N-methyl-N′-[4-(2-{3-[(2-{[tris(propan-2-yl)silyl]oxy}phenyl)methoxy]propyl}phenyl)butyl]ethanediamide (154 mg, crude IK-0357/19) was dissolved in THF (5.00 mL). TBAF*3H 2O (87.0 mg) was added and the resulting mixture was stirred at r.t. for 30 min. Control by LC/MS showed complete conversion. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 75 mg (66%) white solid.

Compound 28 (Comp-28) N′-[(5Z)-13-(2-benzenesulfonamidoethoxy)tridec-5-en-1-yl]-Nmethylethanediamide

N′-[(5Z)-13-(2-aminoethoxy)tridec-5-en-1-yl]-Nmethylethanediamide (BB-11, 50.0 mg, crude IK-0355/8) was suspended in CH2Cl2 (3.00 mL). Benzenesulfonyl chloride (37.5 μL) and NEt 3 (61.1 μL) were added and the reaction mixture stirred at r.t. for 2 h. Control by LC/MS showed product. Control by LC/MS showed product. Control by LC/MS showed product.

The reaction mixture was diluted with water (20 mL) and sat. NaHCO 3 (20 mL) and then extracted with CH 2Cl2 (3×20 mL). The combined organic layers were washed with 1 N HCl (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 42 mg (60%), white solid.

Compound 29 (Comp-29) N′-[(5Z)-13-(2-hydroxyphenoxy)tridec-5-en-1-yl]-Nmethylethanediamide

Step 1:

N′-[(5Z)-13-hydroxytridec-5-en-1-yl]-N-methylethanediamide (BB-8, 400 mg) was suspended in CH 2Cl2 (20 mL). PPh 3 (598 mg) and CBr4 (756 mg) were added and the resulting mixture was stirred at r.t. for 1.5 h. Control by TLC (PE/EtOAc=1:1) and LC/MS showed complete conversion.

The reaction mixture was concentrated in vacuo and the residue was purified by preparative TLC (PE/EtOAc=1:1).

Step 2:

N′-[(5Z)-13-bromotridec-5-en-1-yl]-N-methylethanediamide (50 mg), pyrocatechol (76.2 mg) and K2CO3 (57.4 mg) were placed in a G16 vial. DMF (3.00 mL) was added and the resulting mixture was stirred at 60° C. for 2.5 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (40 mL) and extracted with MTBE (3×20 mL). The combined organic layers were washed with water (20 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 43 mg (80%), white solid.

Compound 30 (Comp-30) N-Methyl-N′-[4-(2-{3-[2-(morpholin-4-yl)-2-oxoethoxy]propyl}phenyl)butyl] ethanediamide

50 mg (0.14 mmol) 2-[3-(2-{4-[(Methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetic acid (BB-8, IK-0358/6), 32.8 mg (0.17 mmol) EDCI and 24.9 mg (0.29 mmol, 24.9 μl) morpholine were dissolved in 3 ml DCM and stirred at rt over the weekend (clear solution).

The mixture was evaporated to dryness and purified via pTLC (1 mm, DCM/MeOH 10:1, Rf of possible product: 0.48).

Yield: 34 mg (58%), white solid.

Compound 31 (Comp-31) N′-(4-{2-[3-(3-hydroxyphenoxy)propyl]phenyl}butyl)-Nmethylethanediamide

Step 1:

N′-{4-[2-(3-hydroxypropyl)phenyl]butyl}-N-methylethanediamide (BB-9, 400 mg) was suspended in CH2Cl2 (20 mL). PPh3 (610 mg) and CBr4 (771 mg) were added and the resulting mixture was stirred at r.t. for 30 min. Control by TLC (PE/EtOAc=1:1) and LC/MS showed complete conversion. The reaction mixture was concentrated in vacuo and the residue was purified by preparative TLC (PE/EtOAc=1:1).

Step 2:

N′-{4-[2-(3-bromopropyl)phenyl]butyl}-N-methylethanediamide (50 mg), 1,3-Benzenediol (77.5 mg) and K2CO3 (58.4 mg) were placed in a G16 vial. DMF (3.00 mL) was added and the resulting mixture was stirred at 60° C. for 1 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (40 mL) and extracted with MTBE (3×20 mL). The combined organic layers were washed with water (20 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 40 mg (74%), white solid.

Compound 32 (Comp-32) N′-[(5Z)-13-(4-hydroxyphenoxy)tridec-5-en-1-yl]-Nmethylethanediamide

Step 1:

N′-[(5Z)-13-hydroxytridec-5-en-1-yl]-N-methylethanediamide (BB-8, 400 mg) was suspended in CH 2Cl2 (20 mL). PPh 3 (598 mg) and CBr4 (756 mg) were added and the resulting mixture was stirred at r.t. for 1.5 h. Control by TLC (PE/EtOAc=1:1) and LC/MS showed complete conversion.

The reaction mixture was concentrated in vacuo and the residue was purified by preparative TLC (PE/EtOAc=1:1).

Step 2:

N′-[(5Z)-13-bromotridec-5-en-1-yl]-N-methylethanediamide (50 mg), hydroquinone (76.2 mg) and

K2CO3 (57.4 mg) were placed in a G16 vial. DMF (3.00 mL) was added and the resulting mixture was stirred at 60° C. for 2.5 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (40 mL) and extracted with MTBE (3×20 mL). The combined organic layers were washed with water (20 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 43 mg (80%), white solid

Compound 33 (Comp-33) N′-(4-{2-[3-(4-hydroxyphenoxy)propyl]phenyl}butyl)-Nmethylethanediamide

Step 1:

N′-{4-[2-(3-hydroxypropyl)phenyl]butyl}-N-methylethanediamide (BB-9, 400 mg) was suspended in CH2Cl2 (20 mL). PPh3 (610 mg) and CBr4 (771 mg) were added and the resulting mixture was stirred at r.t. for 30 min. Control by TLC (PE/EtOAc=1:1) and LC/MS showed complete conversion.

The reaction mixture was concentrated in vacuo and the residue was purified by preparative TLC (PE/EtOAc=1:1).

Step 2:

N′-{4-[2-(3-bromopropyl)phenyl]butyl}-N-methylethanediamide (50 mg), Hydroquinone (77.5 mg) and K2CO3 (58.4 mg) were placed in a G16 vial. DMF (3.00 mL) was added and the resulting mixture was stirred at 60° C. for 1 h. Control by LC/MS showed product.

The reaction mixture was diluted with water (40 mL) and extracted with MTBE (3×20 mL). The combined organic layers were washed with water (20 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 39 mg (72%), white solid

Compound 34 (Comp-34) N′-[4-(2-{3-[(4-hydroxyphenyl)methoxy]propyl}phenyl)butyl]-N-methylethanediamide

Step 1

4-Hydroxybenzaldehyde (2.00 g) and imidazole (2.79 g) were dissolved in DMF (20.0 mL). TIPSCl (5.96 mL) was added and the resulting mixture was stirred at 60° C. for 2d.

Control by TLC (PE/EtOAc=95:5) and LC/MS showed complete conversion.

The reaction mixture was diluted with water (100 mL) and extracted with MTBE (3×40 mL). The combined organic layers were washed with 1N NaOH (30 mL) and brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=95:5).

Yield: 3.94 g (86%) pale yellow oil

Step 2

4-{[tris(propan-2-yl)silyl]oxy}benzaldehyde (3.94 g) was dissolved in EtOH (30.0 mL) and cooled to 0° C. NaBH 4 (535 mg) was added and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 18 h.

Control by TLC (PE/EtOAc=8:2) and LC/MS showed product.

The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2 (PE/EtOAc=8:2).

Step 3

(4-{[tris(propan-2-yl)silyl]oxy}phenyl)methanol (300 mg) was dissolved in dry THF (5.00 mL). NaH (60% in mineral oil, 64.2 mg) was added and the mixture stirred at r.t. for 15 min. Allylbromide (231 μL) was then added and the resulting mixture was stirred at r.t. for 2.5 h. Control by LC/MS showed complete conversion.

The reaction mixture was poured into water (30 mL) and extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was used for further transformation as such.

Yield: 372 mg (crude) yellow oil

Step 4

{4-[(prop-2-en-1-yloxy)methyl]phenoxy}tris(propan-2-yl)silane (356 mg) was placed in a 10 mL flask and cooled to 0° C. under and Ar-atmosphere. 9-BBN (0.5 M in THF, 3.33 mL) was then added dropwise and the resulting mixture was stirred at 0° C. for 30 min and then at r.t. for 2 h.

A solution of Na2CO3 (147 mg) in water (3.00 mL) was then added and stirring was continued at r.t. for 30 min. Then N′-[4-(2-iodophenyl)butyl]-N-methylethanediamide (BB-2, 100 mg) and PdCl2(PPh3)2 (9.7 mg) were added and the mixture heated to 50° C. for 2 h. Control by LC/MS showed product. The reaction mixture was cooled to r.t., diluted with water (20 mL) and the layers were separated. The aqueous layer was extracted with EtOAc (15 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was passed through a short column of SiO2 (PE/EtOAc 1:1). The still crude product was then used for further transformation as such.

Yield: 248 mg (crude) yellow oil.

Step 5

N-methyl-N′-[4-(2-{3-[(4-{[tris(propan-2-yl)silyl]oxy}phenyl)methoxy]propyl}phenyl)butyl]ethanediamide (154 mg, crude IK-0357/20) was dissolved in THF (5.00 mL). TBAF*3H 2O (131 mg) was added and the resulting mixture was stirred at r.t. for 30 min. Control by LC/MS showed complete conversion.

The reaction mixture was poured into water (40 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 76 mg (68%), white solid.

Compound 35 (Comp-35) N′-[(5Z)-13-[(methanesulfonylcarbamoyl)methoxy]tridec-5-en-1-yl]-Nmethylethanediamide

2-{[(8Z)-13-[(methylcarbamoyl)formamido]tridec-8-en-1-yl]oxy}acetic acid (BB-4, 50.0 mg) was dissolved in THF (2.00 mL). 1,1′-Carbonyldiimidazole (25.0 mg) was added and the resulting mixture was stirred at r.t. for 1.5 h. Then DBU (50.0 μL) and methanesulphonamide (16.0 mg) were added and stirring was continued at r.t. for 18 h. Control by LC/MS showed product. The reaction mixture was diluted with water (30 mL) and extracted with CH 2Cl2 (3×20 mL). The combined organic layers were washed with 1N HCl (20 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative TLC (CH2Cl2/MeOH=95:5).

Yield: 27 mg (44%) white solid

Analytical Devices Used to Analyze Comp-14 to Comp-34

Analytical LC/ESI-MS: Waters 2700 Autosampler. Waters 1525 Multisolvent Delivery System. 5 μL sample loop. Column, Phenomenex Onyx Monolythic C18 50×2 mm, with stainless steel 2 μm prefilter. Eluent A, H₂O+0.1% HCOOH; eluent B, MeCN. Gradient, 5% B to 100% B within 3.80 min, then isocratic for 0.20 min, then back to 5% B within 0.07 min, then isocratic for 0.23 min; flow, 0.6 ml/min and 1.2 ml/min.

Waters Micromass ZQ 4000 single quadrupol mass spectrometer with electrospray source. MS method, MS4_15 minPM-80-800-35V; positive/negative ion mode scanning, m/z 80-800 in 0.5 s; capillary voltage, 3.50 kV; cone voltage, 50 V; multiplier voltage, 650 V; source block and desolvation gas temperature, 120° C. and 300° C., respectively. Waters 2487 Dual A Absorbance Detector, set to 254 nm. Software, Waters Masslynx V 4.0.

Waters Micromass LCZ Platform 4000 single quadrupol mass spectrometer with electrospray source. MS method, MS4_15 minPM-80-800-35V; positive/negative ion mode scanning, m/z 80-800 in 1 s; capillary voltage, 4.0 kV; cone voltage, 30 V; multiplier voltage, 900 V; source block and desolvation gas temperature, 120° C. and 300° C., respectively. Waters 996 Photodiode Array Detector, set 200 to 400 nm. Software, Waters Masslynx V4.0.

Values for [M+H]+ given in the examples are those found within the corresponding LC/MS chromatogram for the respective compound. These values were all found within tolerable margins of +/−0.3 units compared to calculated exact mass upon protonation of the compound.

Preparative thinlayer chromatography (preparative TLC): Merck PLC plates, silica gel 60 F254, 0.5 mm, 1.0 mm or 2.0 mm.

Column chromatography: Acros silica gel 60A, 0.035-0.070 mm.

Preparative HPLC-MS: Waters 2767 Autosampler, Waters 600 Multisolvent Delivery System with analytical pump heads (100 μL); Waters 600 Controller; Waters 2525 Binary Gradient Modul with preparative pump heads (500 μL). At-Column-Dilution: solvent1, MeCN:H2O 70:30 (v/v), solvent2, MeCN:MeOH:DMF 80:15:5 (v/v/v); flow rate, 5 mL/min. Autosampler 2767 with 10 mL syringe and 10 mL Sample loop. Column 6-position valve Flom 401 with Waters X-Terra RP18, 5 μm, 19×150 mm with X-Terra RP18 guard cartridge 5 μm, 19×10 mm, used at flow rate 20 mL/min; Waters SunFire Prep OBD 5 30×50 mm with SunFire RP18 guard cartridge 5 μm, 19×10 mm, used at flow rate 25 mL/min; Waters Atlantis Prep T3 OBD 5 μm, 30×50 mm with Atlantis guard cartridge, used at flow rate 50 mL/min; Waters X-Bridge Prep OBD 5 μm, 19×150 mm with X-Bridge RP18 guard cartridge 5 μm, 19×10 mm used at flow rate 20 mL/min; Waters Atlantis Prep T3 OBD 5 μm, 19×50 mm with Atlantis guard cartridge, used at flow rate 25 mL/min and YMC-Actus Hydrosphere C18 5 μm, 20×50 mm with Actus guard cartridge, used at flow rate 20 mL/min. Eluent A, H₂O containing 0.1% (v/v) HCOOH or H₂O containing 0.1% (v/v) NEt3; eluent B, MeCN. Different linear gradients, individually adapted to sample. Injection volume, 9 mL, depending on sample. Make-up solvent, MeOH-MeCN—H2O—HCOOH 80:15:4.95:0.05 (v/v/v/v). Make-up pump, Waters Reagent Manager, flow rate 0.5 mL/min. Waters ZQ single quadrupole mass spectrometer with electrospray source. Positive or negative ion mode scanning m/z 105-950 in 1 s; capillary, 3.6 kV; cone voltage, 45 V; multiplier voltage, 700 V; probe and desolvation gas temperature, 120° C. and 250° C., respectively. Waters Fraction Collector 2767 with mass or UV-triggered fraction collection. Waters 2487 Dual A Absorbance Detector, set to 254 nm. Software, Waters Masslynx V 4.0 SP4.

1H NMR spectra were recorded at room temperature on a Bruker Supraleitendes Fourier NMR Spektrometer, Avance™ 300 MHz. Chemical shifts 6 are reported in ppm. Multiplicity of a certain signal (singlet, doublet, triplet, quartet, multiplet) is indicated by the respective abbreviation (s, d, t, q, m respectively). “br s” indicates a broad singlet, “mC” a centered multiplet. The solvent residual signals were used as internal standards: δ(CDCl3)=7.26, δ(d6-DMSO)=2.50, δ(CD3OD)=3.31, δ(d6-acetone)=2.05.

TABLE xxx Calculated exact mass of Comp-14 to Comp-34 Calculated Compound IUPAC name Comp-00 exact mass M + 1 N-methyl-N′-[(5Z)-13-[(1H-1,2,3,4-tetrazol-5-yl)methoxy]tridec-5-en-1- Comp-14 380.25359 381.260869 yl]ethanediamide N′-(4-{2-[3-(Carbamoylmethoxy)propyl]phenyl}butyl)-N-methylethanediamide Comp-15 349.20016 350.207437 N-Methyl-N′-[(5Z)-13-[(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3- Comp-16 395.25326 396.260535 yl)methoxy]tridec-5-en-1-yl]ethanediamide N-methyl-N′-[(5Z)-13-[(phenylcarbamoyl)methoxy]tridec-5-en-1- Comp-17 431.27841 432.285687 yl]ethanediamide N-methyl-N′-[(5Z)-13-{[(oxan-4-yl)carbamoyl]methoxy}tridec-5-en-1- Comp-18 439.30462 440.311902 yl]ethanediamide N-methyl-N′-[(5Z)-13-{[(1,3-oxazol-2-yl)carbamoyl]methoxy}tridec-5-en-1- Comp-19 422.25292 423.260201 yl]ethanediamide N′-[(5Z)-13-{[(4-methoxyphenyl)carbamoyl]methoxy}tridec-5-en-1-yl]-N- Comp-20 461.28897 462.296252 methylethanediamide N-Methyl-N′-[4-(2-{3- Comp-21 425.23146 426.238737 [(phenylcarbamoyl)methoxy]propyl}phenyl)butyl]ethanediamide N-Methyl-N′-[4-(2-{3-[2-oxo-2-(pyrrolidin-1- Comp-22 403.24711 404.254387 yl)ethoxy]propyl}phenyl)butyl]ethanediamide N-Methyl-N′-[4-(2-{3-[2-(morpholin-4-yl)-2- Comp-23 419.24202 420.249302 oxoethoxy]propyl}phenyl)butyl]ethanediamide N′-[(5Z)-13-{[(benzenesulfonyl)carbamoyl]methoxy}tridec-5-en-1-yl]-N- Comp-24 495.24031 496.247589 methylethanediamide 4-{2-[3-(2-{4- Comp-25 469.22129 470.228567 [(methylcarbamoyl)formamido]butyl}phenyl)propoxy]acetamido}benzoic acid N′-[(5Z)-13-[2-(4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)-2-oxoethoxy]tridec-5- Comp-26 438.2366 439.243883 en-1-yl]-N-methylethanediamide N′-[4-(2-{3-[2-(hydroxymethyl)phenoxy]propyl}phenyl)butyl]-N- Comp-27 398.22056 399.227838 methylethanediamide N′-[(5Z)-13-(2-benzenesulfonamidoethoxy)tridec-5-en-1-yl]-N- Comp-28 481.26104 482.268324 methylethanediamide N′-[(5Z)-13-(2-hydroxyphenoxy)tridec-5-en-1-yl]-N-methylethanediamide Comp-29 390.25186 391.259138 N′-[(5Z)-13-(3-hydroxyphenoxy)tridec-5-en-1-yl]-N-methylethanediamide Comp-30 390.25186 391.259138 N′-(4-{2-[3-(3-hydroxyphenoxy)propyl]phenyl}butyl)-N-methylethanediamide Comp-31 384.20491 385.212188 N′-[(5Z)-13-(4-hydroxyphenoxy)tridec-5-en-1-yl]-N-methylethanediamide Comp-32 390.25186 391.259138 N′-(4-{2-[3-(4-hydroxyphenoxy)propyl]phenyl}butyl)-N-methylethanediamide Comp-33 384.20491 385.212188 N′-[4-(2-{3-[(4-hydroxyphenyl)methoxy]propyl}phenyl)butyl]-N- Comp-34 398.22056 399.227838 methylethanediamide N′-[(5Z)-13-[(methanesulfonylcarbamoyl)methoxy]tridec-5-en-1-yl]-N- Comp-35 433.22466 434.231939 methylethanediamide

EXAMPLE 2: ANTI-ARRHYTHMIC EFFECT OF METABOLICALLY ROBUST ANALOGS OF 17,18-EEQ ON NRCMS

Materials and Methods

The structures of all compounds tested are given in FIG. 1. The compounds include analogues being part of the invention (Comp-01 to Comp-05), which were synthesized as described in example 1, and additional related compounds (Comp-06 to Comp-13). A 1000-fold stock solutions in ethanol of the test compounds was prepared before use.

In order to measure the biological activities of metabolically robust analogs of CYP-eicosanoids an established cell model was used (Kang, J. X. et al., Proc Natl Acad Sci USA, 1994. 91(21): p. 9886-90). The spontaneously beating neonatal rat cardiomyocytes (NRCMs) are a model system to investigate anti-arrhythmic effects of test-compounds. Indicating their anti-arrhythmic potential, test compounds reduce the spontaneous beating rate of these cells and prevent their irregular and asynchronous contraction in response to arrhythmic substances.

Isolation and cultivation of NRCMs were performed as described previously (Wallukat, G et al., J Clin Invest. 1999; 103: 945-952; Falck, J R et al., J Med Chem. 2011 Jun. 23; 54(12):4109-18). The isolated cells were cultured as monolayers on the bottom (12.5 cm2) of Falcon flasks in 2.5 ml of Halle SM 20-l medium equilibrated with humidified air. The medium contained 10% heat-inactivated FCS and 2 μmol/l fluoro-deoxyuridine (Serva, Heidelberg, Germany), the latter to prevent proliferation of non-muscle cells. The NRCMs (2.4×106 cells/flask) were cultured at 37° C. in an incubator. After 5 to 7 days, the NRCMs formed spontaneously beating cell clusters. The cells in each cluster showed synchronized contraction with a beating rate of 120 to 140 beats per minute. On the day of the experiment, the culture medium was replaced by 2.0 ml fresh serum-containing medium. After four hours of incubation cells were adopted for 10 min to 31° C. and beating was recorded using an inverted microscope (Leica DM IRB) equipped with a CCD camera and coupled to an IonOptix (software: IonWizard6, IonOptix). To determine the basal rate, 6 to 8 individual clusters were selected and the number of contractions was counted for 15 sec. After that, the compound to be tested was added to the culture and the beating rate of the same clusters was monitored 5 min later again. Based on the difference between the basal and compound-induced beating rate of the individual clusters, the chronotropic effects (Δ beats/min) were calculated and are given as mean±/−SE values. N refers to the number of clusters monitored which originated, in general, from at least three independent NRCM cultures. Stock solutions of the compounds were prepared in ethanol and applied to give a final concentration of either 20 nM or 30 nM on NRCMs (n=6 per compound). The vehicle control (0.1%) showed no effect on the basal beating rate.

Results

The results are presented in FIG. 1. The potential of the 17,18-EEQ analogs to reduce the spontaneous beating rate ranged between −1.3+/−1.0 delta bpm up to −38.0+/−3.3 delta bpm according to distinct structural features. Free carboxylic acid derivatives showed a greater reduction than analogs where the carboxy group was esterified to a polyalkoxyalkyl or an amino acid (Comp-10 vs. Comp-07 and Comp-08 and Comp-06 vs. Comp-09). The results also showed, that it is possible to replace the double bond in 11,12-position with a phenyl ring without major loss of activity (Comp-06 and Comp-05). However, shifting the 11,12-double bond to the 14,15-position strongly reduced the negative chronotropic effect on NRCMs (Comp-06 vs. Comp-11). Moreover, analogs containing an 3-oxa group showed an equal potency to reduce the beating rate (Comp-06 vs. Comp-02 and Comp-03 or Comp-10 vs. Comp-01) or increased the negative chronotropic effect (Comp-10 vs. Comp-04). For the oxamide group it was shown that it was essential for the in vitro efficacy, since two degradation products of the oxamide group were inactive (Comp-06 vs. Comp-12 and Comp-13). As shown in FIG. 1 continued further compounds bearing both an 3-oxa and an oxamide group showed good activity (Comp-14 to Comp-35).

EXAMPLE 3: ANTI-ARRHYTHMIC EFFECT OF 17,18-EEQ AGONISTS COMP-02 ON ATRIAL FIBRILLATION

This example shows that the agonistic analog Comp-02 ameliorates atrial fibrillation

Materials and Methods

Study design: To gain insight into in-vivo effect of synthetic 17,18-EEQ-agonists, atrial fibrillation studies were performed in male B16 mice as described in Westphal, C. et al., PLoS ONE. 2013, 8(8): e73490. Briefly, moderate cardiac hypertrophy was induced by continuous infusion of isoproterenol via subcutaneously implanted osmotic minipumps (Alzet) at a rate of 40 mg/kg/d for two weeks. After two weeks of treatment, ECG and electrophysiological data were recorded. Programmed electrical stimulation (PES) was performed in the right atrium or right ventricle using a digital electrophysiology lab (EP Tracer; CardioTek) to determine refractory periods and arrhythmia inducibility. Atrial arrhythmias were defined as fast (>800 bpm) electrical activity in the right atrial electrograms, with ECG P waves different to normal sinus rhythm and subsequent fast, but physiological activation of the ventricles (ECG R wave and right ventricular electrograms similar to normal sinus rhythm). Atrial fibrillation was defined as fast, irregular activity in the right atrial electrograms with irregular conduction to the ventricles (high variability of R—R intervals). Ventricular arrhythmias were defined by fast (>800 bpm) activity originating from the ventricular myocardium (change in morphology of ECG R waves and local right ventricular electrograms compared to normal sinus rhythm). During inhalation anesthesia with isoflurane (2% with 360 ml/min air flow; Univentor 400 anesthesia unit), the animals' body temperature was kept constant at 37° C. using a homeothermic blanket control unit (Hugo Sachs Elektronik, Harvard Apparatus) with rectal temperature control. After preparation of the right jugular vein, a 2 French octapolar electrophysiology catheter (CIB'ER mouse cath; NuMed) was placed in the right heart, including atrium and ventricle. PES was performed using a standardized protocol that included trains of 10 basal stimuli (S1) followed by up to 3 extra stimuli (S2-S4), delivered with a coupling interval decreasing in steps of 5 ms until ventricular or atrial refractoriness was reached. The stimulation procedures were repeated at three different basal cycle lengths (100 ms, 90 ms, 80 ms) with each animal. Occurrence and duration of inducible arrhythmias were documented. Only stimulation protocols with reproducible arrhythmias longer than five consecutive beats in ventricle and episodes longer than 350 ms in the atria were considered positive. “Arrhythmia inducibility” was calculated as the percentage of effective (positive) out of total protocols applied. Accordingly, the arrhythmia inducibility of individual animals could take a value of 0, 33, 66 or 100%. For statistical evaluation, the data obtained for the individual animals in a given group were averaged and are given as mean±SEM. For scoring the severity of induced arrhythmias, three response categories were defined: sustained (≥10 consecutive ventricular extrasystoles, VES or atrial fibrillation episodes ≥30 sec in at least one protocol), non-sustained (<10 VES or atrial fibrillation episodes <30 sec in at least one protocol) and no arrhythmias in all three protocols. The data are given as percentage of animals in a given group assigned to these categories.

Results

The results are presented in FIG. 2. Bolus injection of the synthetic 17,18-EEQ agonist (Comp. 02) did not induce any obvious negative side effects. Programmed electrical stimulation induced atrial fibrillation in a majority of vehicle-treated mice (n=12). A single i.v. injection of the synthetic 17,18-EEQ-agonist Comp-02 (2 mg/kg body weight) reduced significantly the sum of totally induced atrial fibrillation episodes (atrial fibrillation burden) (n=14); FIG. 2 A. Moreover, the severity of induced atrial fibrillation episodes was significantly reduced. In particular, the inducibility of sustained arrhythmia episodes was significantly reduced by 62%; FIG. 2 B.

EXAMPLE 4: ANTI-ARRHYTHMIC EFFECT OF 17,18-EEQ AGONISTS ON SPONTANEOUSLY VENTRICULAR TACHYCARDIAS IN ACUTE MYOCARDIAL INFARCTION

This example shows that the agonistic analog Comp-03 ameliorates arrhythmias as induced by myocardial infarction.

Materials and Methods

Study design: To gain insight into the in-vivo effects of synthetic 17,18-EEQ-agonists, myocardial infarction studies were performed in male Wistar rats. Briefly, rats weighing 220-250 g were randomized to receive an i.v. bolus of Comp-03 (100 μg in 300 μl 0.9% NaCl) or only 300 μl 0.9% NaCl as vehicle control ten minutes before induction of myocardial infarction. For safe bolus application, animals were mildly anesthetized using isoflorane. Continuous monitoring of the surface-ECG was started (EPTracer, Netherlands) and maintained until the end of the study (45 minutes after induction of myocardial infarction). After recording of the basal ECG, myocardial infarction was induced by ligation of the left anterior descending artery (LAD). 45 minutes after myocardial infarction animals were sacrificed and organ harvested. Samples from urine, blood, liver, kidney and heart were stored for further analysis.

Method of arrhythmia analysis: Ventricular tachycardia burden was calculated as the sum of all arrhythmic events originating from the ventricular myocardium, which were observed within the first hour after induction of myocardial infarction. In order to quantify not only the frequency but also the severity of the ventricular arrhythmias, an arrhythmia-severity-score was calculated. This score was calculated as the sum of the number of different arrhythmia events (PVC, couplet, triplet, VT<1.5 sec, VT >=1.5 sec), each class factorized by an increasing severity index of 1-5 (e.g. PVC×1, couplets×2, triplets 1.5 sec×3, VT>=1.5 sec×5).

Results

The results are presented in FIG. 3. Bolus injection of the synthetic 17,18-EEQ agonist (Comp-03) did not induce any obvious negative side effects. Ventricular arrhythmias occurred after coronary artery ligation and were observed as single premature ventricular contractions (PVC), short runs of non-sustained ventricular tachycardia (VT) and ventricular tachycardia/fibrillation. Rats treated with the synthetic 17,18-EEQ-agonist (n=10) showed a significantly reduced ventricular tachycardia duration compared to controls (n=9); FIG. 3 A. Moreover, the arrhythmia severity score was significantly reduced. FIG. 3 B.

EXAMPLE 5: CARDIOPROTECTIVE EFFECT OF 17,18-EEQ IN ISCHEMIA/REPERFUSION DAMAGE OF THE HEART

This example shows that the agonistic analog Comp-03 ameliorates post-ischemic recovery as induced by a defined period of ischemia.

Materials and Methods:

Study design: To gain insight into the cardioprotective effects of synthetic 17,18-EEQ-agonists, ischemia-reperfusion studies were performed in isolated mouse hearts. Briefly, hearts were perfused in the Langendorff mode as described in Seubert et al., Circ Res. 2004; 95:506-514. Hearts were perfused with buffer for a 10-minutes stabilization period, then perfused with either the agonistic analog Comp-03 (1 μM) or vehicle for 10 minutes, then subjected to 35 minutes global no-flow ischemia, followed by 40 minutes reperfusion. During ischemia and reperfusion the infusion with either the agonistic analog Comp-03 or vehicle was kept constant. Recovery of contractile function was taken as left ventricular developed pressure (LVDP) at the end of reperfusion expressed as a percentage of pre-ischemic LVDP.

Results

The results are presented in FIG. 4. Continuous infusion of 1 μM of the synthetic 17,18-EEQ agonist (Comp-03) did not induce any obvious negative side effects. Immediately after the 35 minutes episode of global ischemia contractility of the control hearts (n=14) as expressed by the left ventricular developed pressure (LVdP) were strongly reduced and returned then gradually in the reperfusion phase to about 50% of the pre-ischemic values. Hearts treated with the synthetic 17,18-EEQ-agonist Comp-03 (n=14) showed significantly improved recovery of contractility.

EXAMPLE 6: IN VITRO INHIBITION OF RECOMBINANT HUMAN SOLUBLE EPOXIDE HYDROLASE

Materials and Methods

A selection of compounds was tested for the ability to inhibit human soluble expoxide hydrolase (sEH). The metabolically robust 17,18-EEQ analogs themselves are not prone to metabolization by the sEH but might act as inhibitors of this enzyme. Briefly, the assay was performed at 37° C. for 20 min in a final volume of 100 μL potassium phosphate buffer (0.1 M, pH 7.2) containing 50 μM 14,15-EET as substrate. The reactions were started by adding enzyme (107.5 ng per reaction, 12.06 U/ml activity, Cayman Chemicals) and terminated with 300 μl ethyl acetate. The remaining substrate and its product (14,15-DHET) were extracted and analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) (Muller, D. N. et al., Biochem J 2007. 403(1): p. 109-118). Metabolically robust analogs shown in FIG. 5 were tested at final concentration of 10 μM for potency of sEH-inhibition compared to vehicle control (1% DMSO), n=2-4.

Results

FIG. 5 shows that some of the tested 17,18-EEQ analogs inhibited human sEH up to 76.6%. These compounds share one structural feature, containing an urea group (Comp-01, -07, -08 and Comp-10). In contrast, both compounds tested containing an oxamide group did not show sEH-inhibition (Comp-02 and Comp-03).

EXAMPLE 7: PERMEABILITY POTENTIAL OF METABOLICALLY ROBUST ANALOGS OF CYP-EICOSANOIDS TESTED IN CACO-2 CELLS

Materials and Methods

To predict human intestinal permeability and to investigate drug efflux, parameters affecting the bioavailability of a compound (van Breemen R B & Li Y., Expert Opin Drug Metab Toxicol. 2005 August; 1(2):175-85.), a selection of metabolically robust 17,18-EEQ analogs were tested for their potential to permeate through a confluent monolayer of Caco-2 cells (a human colon adeno-carcinoma cell line). Stock solutions of compounds were generated in DMSO (1 mM) and compounds were tested at a final concentration of 1 μM. A solution of a test compound placed on the apical side of a Caco-2 cell monolayer (2 h incubation time), and the rates of appearance of the test compounds on the basolateral side of the cells are measured to assess the permeability of the monolayer and are given as permeability from A to B in cm/s for the tested compounds (FIG. 6).

Results

The results presented in FIG. 6 show that compounds with an urea group showed less permeability (Comp-01 and Comp-04) than compounds containing an oxamide group (Comp-02 and Comp-03). An additional polyalkoxyalkyl esterified to the carboxylic acid group further improved permeability (Comp-02 vs. Comp-03).

EXAMPLE 8: ABILITY OF METABOLICALLY ROBUST ANALOGS OF CYP-EICOSANOIDS TO INCORPORATE INTO PHOSPHOLIPID MEMBRANES

Material and Methods

To see if modified structures (selected metabolically robust analogs of CYP-eicosanoids, Table 1) are able to incorporate into phospholipid membranes, a cardiomyocyte cell line (H9c2, rat) was incubated with test compounds for four hours at a final concentration of 1 μM (n=2). After incubation cellular lipids were extracted with a chloroform/methanol 1:2 mixture. To differentiate between free and incorporated compounds an aliquot of the samples was subjected to alkaline hydrolysis. Both extracts were analyzed for the test compounds using LC-MS/MS. 17,18-EEQ and 20-HETE (Kaduce T L et al., J Biol Chem. 2004 Jan. 23; 279(4):2648-56.) served as positive control for membrane incorporation.

Results

FIG. 7 shows that in contrast to the positive controls (17,18-EEQ and 20-HETE) the compounds of invention (Comp-02 and Comp-04) as well as the oxamide containing compound (Comp-06) showed no incorporation into phospholipid membranes. The urea Comp-10 lacking the 3-oxa group showed weak incorporation.

EXAMPLE 9: SOLUBILITY ASSESSMENT OF METABOLICALLY ROBUST ANALOGS OF 17,18-EEQ

Materials and Methods

Aqueous solubility of Comp-06 and Comp-02 was determined in deionised water at 37° C. Known amounts of the compounds were suspended by agitation for 24 hours. The filtrates were isolated and content of the appropriate compounds was determined by HPLC analysis.

Results

In Table 1 it is shown, that the introduction of an 3-oxa group into the structure improved solubility. Comparing the solubility in deionised water, solubility of Comp-02 was approximately ×10 greater than that of Comp-06.

TABLE 1 OMT-02 OMT-06 Highest and Average OMT-02 Highest and lowest Average OMT-06 Aqueous lowest OMT-02 solubility at OMT-06 solubility solubility at media, solubility values 37° C., values at 37° C., 37° C., 0.1M at 37° C., mg/ml mg/ml mg/ml mg/ml Deionised 0.075 0.068 0.0070 0.0068 water, pH 0.059 0.0066 5.90

EXAMPLE 10: PHARMACOKINETIC PROPERTIES OF METABOLICALLY ROBUST ANALOGS OF 17,18-EEQ

Material and Methods

Study Design:

To gain insight into pharmacological properties of synthetic 17,18-EEQ agonists pharmacokinetic studies were performed with two selected compounds (Comp-02 and Comp-06). These compounds were pharmacologically evaluated in plasma after single intravenous and oral administration. Therefore, male C57BL/6 mice (Janvier Labs (France), n=12 per group) were administered to the test item at a dose of 2 mg/kg (i.v.) and 8 mg/kg (p.o.), respectively. Intravenous application of Comp-02 (sodium salt in isotonic salt solution) and Comp-06 (free acid in DMSO/PEG400 20:80) was performed via the tail vein, and for oral administration via gavage. To assure equal solubility Comp-06 was formulated in DMSO/PEG400 20:80, since Comp-06 showed impaired solubility as sodium salt. Blood sampling was of 100 μl were obtained at two different time point for each mouse (0.083/2 h, 0.25/4 h, 0.5/8 h and 1/24 h). Biological samples were subjected to an extraction procedure (40 μl Acetonitril+22.4 μl sample, shaking, 10 min centrifugation at 6000×g at room temperature, dilution of 50 μl supernatant 1:1 with water). For analysis 20 μl of sample were injected into the LC-MS, Accela 1250 UHPLC system, Accela Open Autosampler, Q-Exactive mass spectrometer (Thermo Fisher Scientific). Appropriate samples were used as controls: zero sample, calibration standard and QC sample. The pharmacokinetic analysis were performed applying a non-compartment model using the Kinetica 5.0 software (Thermo Scientific, Waltham, USA). All given parameters were obtained by trapezoid area calculation.

Cmax (ng/ml) maximal observed plasma concentration tmax (h) time of maximal observed plasma concentration AUC0-∞(ng*h/ml) area under the concentration-time curve extrapolated to infinity F (%) oral bioavailability expressed as percentage Results

The results presented in Table 2 show improved properties in pharmacokinetic parameters of Comp-02 compared to Comp-06. The compound of invention (Comp-02) differs only in an additional 3-oxa group, which is missing in Comp-06. This structural feature greatly improved the oral bioavailability (87% vs 5.3%). Moreover, the maximal observed concentration was much higher and reached faster with Comp-02 (903.3 ng/ml and 0.3 h) than Comp-06 (24.4 ng/ml and 0.5 h). Further, the AUC0-∞, as measure for total compound exposure over time, was higher with Comp-02 (1818 ng*h/ml) compared to Comp-06 (31 ng*h/ml).

TABLE 2 Comp-02 Comp-06 Iv po iv po 2 mg/kg 8 mg/kg 2 mg/kg 8 mg/kg C max (ng/ml) — 903.8 — 24.4 t max (h) — 0.3 — 0.5 AUC0-∞(ng*h/ml) 524 1818 146 31 F (%) 100 87 100 5.3

EXAMPLE 11: CARDIOPROTECTIVE EFFECT OF SYNTHETIC 17,18-EEQ-AGONIST IN ISCHEMIA/REPERFUSION OF THE HEART

Further to the example 5, this example shows that another agonistic analog of the invention, namely Comp-02 ameliorates post-ischemic recovery as induced by a defined period of ischemia.

Materials and Methods

Study design: To gain insight into the cardioprotective effects of synthetic 17,18-EEQ-agonists, ischemia-reperfusion studies were performed in isolated mouse hearts of 12-14 week old male C57BL6/n animals. Briefly, mice were anesthetized with i.p. injected pentobarbital/heparin, hearts were rapidly removed and perfused in a Langendorff apparatus. Excised hearts were put in ice-cold Krebs-Henseleit buffer and aortas were cannulated. Modified Krebs-Henseleit buffer for retrograde perfusion contained 118 mM NaCl, 3.5 mM KCl, 1.3 mM MgSO₄, 2.5 mM CaCl₂, 24.7 mM NaHCO₃, 1.4 mM KH₂PO₄ and 11 mM glucose, was aerated with 95% O₂-5% CO₂ and kept at constant temperature of 37° C. and pH 7.3. Perfusion pressure was adjusted to 80 mmHg and a latex balloon connected to a pressure transducer was inserted in the left ventricle through the left atrium. Hearts were perfused with buffer for a 15 minutes stabilisation period, followed by 10 minutes perfusion with either the agonistic analog Comp-02 (100 nM) or vehicle (0.9% NaCl in water). Afterwards hearts were subjected to 35 minutes global no-flow ischemia followed by 40 minutes of reperfusion. Compound or vehicle perfusion was kept constant during the reperfusion time. To analyse recovery of contractile function the left ventricular developed pressure (LVdP) at different time points during reperfusion was measured and expressed as percentage of pre-ischemic LVdP. Exclusion criteria for the hearts were: (i) a LVdP below 60 mmHg at the end of the stabilisation phase (baseline measurement), (ii) a heart rate below 260 bpm, (iii) coronary flow below 1.5 or over 4 ml/min and finally (iv) severe sustained arrhythmias during the stabilisation phase. The results are presented in FIG. 1 and expressed as mean±SEM percentage of pre-ischemic LVdP. Data were analysed by two-tailed unpaired Student's t test and considered significant if p<0.05*.

Results

FIG. 8 shows that continuous infusion of 100 nM of Comp-02 did not induce any obvious negative side effects. After global ischemia, contractility of the control hearts (n=5) was strongly reduced. After 10 min of reperfusion time, pre-ischemic LVdP values ware at 1% but returned gradually to about 18% after 40 minutes reperfusion. In contrast, hearts treated with Comp-02 (n=5) showed significantly improved post-ischemic recovery of contractility function compared to the control group. Early (10 minutes) as well as late (40 minutes) time points during reperfusion phase showed better functional recovery with 18% and 59% of the pre-ischemic LVdP values, respectively.

EXAMPLE 12: CARDIOPROTECTIVE EFFECT OF ROBUST ANALOGS OF 17,18-EEQ ON ISOLATED PRIMARY CARDIOMYOCYTES

Materials and Methods

In vitro ischemic injury was induced in mouse neonatal primary cardiomyocyte culture by oxygen-glucose deprivation for 18 hours and a 24 hours reoxygenation period in a humidified incubator with 5% CO₂/95% air. Cardiomyocytes were incubated with the test compounds during 4 hours prior to the oxygen glucose depriviation (OGD) induction. The test compounds were also present during the OGD insult and during the 24 h-reoxygenation period. After the reoxygenation period, cell number was assessed by Hoechst 33342 staining, apoptosis was measured by determining caspase 3/7 activation, and cell membrane integrity or necrosis was measured as LDH release.

Data were expressed as mean±SD of three separate wells and individual comparisons were made with Student's t-test (SigmaPlot 9.0 program). The results were normalized to the normoxia-treated cells. Statistical significance (*) p<0.05 as compared to normoxia/DMSO-treated cells and (§) hypoxia/DMSO treated cells. Results

FIG. 9A to 9B show, that Comp-02 partially protected against OGD-induced damage in primary cardiomyocytes. It ameliorates loss of cell number (by 35% with 30 nM or by 44% with 10 μM) and reduced OGD-induced apoptosis and necrosis (by 43% and 29% respectively). The natural precursor of the compound of invention 17,18-EEQ appeared less efficacious, as it did not significantly affect cell number (at 30 nM and 10 μM), and reduced apoptosis only at high concentration (10 μM, 43%) but not at the low concentration tested (30 nM). However, to prevent OGD-reoxygenation induced cellular necrosis, as tested by LDH-release, Comp-02 and 17,18-EEQ seemed similarly effective. Comp-02 reduced cellular necrosis by 43% (30 nM) and by 45% (10 μM) respectively. Comparably, 17,18-EEQ reduced LDH-release by 41% (30 nM) and by 37% (10 μM) respectively, see FIG. 9C 

The invention claimed is:
 1. A compound of the general formula (I): P-E-I  (I) or a pharmaceutically acceptable salt thereof, wherein P is a group represented by the general formula (II): (CH₂)_(n)—O—(CH₂)_(k)—X  (II) wherein n is an integer of from 3 to 8; and k is 0, 1 or 2; X represents CH₂OH, CH₂OAc, CH(O) or a group selected from the group consisting of:

wherein R and R′ each independently represents a hydrogen atom; or a C₁-C₆alkyl group which may be substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R¹ represents a hydroxyl group, C₁-C₆alkoxy, NHCN, —NH(C₁-C₆alkyl), —NH(C₃-C₆cycloalkyl), —NH(aryl), or —O(C₁-C₆alkyldiyl)O(C═O)R¹¹; R¹¹ is a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s); or a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R² represents —NHR³; —NR²⁰R²¹; —OR²²; —(OCH₂—CH₂)_(i)—R²³; —C₃-C₁₀-heterocyclyl optionally substituted with one, two or three substituents independently selected from the group consisting of hydroxyl group, C₁-C₆alkoxy, C₁-C₆alkyl, and oxo; —(Xaa)_(o); a mono-, or disaccharide, or a derivative thereof, which is joined to —C(O) by an ester bond via the 1-O, 3-O-, or 6-O-position of the saccharide; or is selected from the group consisting of:

wherein R³ represents (SO₂R³⁰); (OR³¹); —C₁-C₆alkanediyl(SO₂R³²); —C₁-C₆alkanediyl(CO₂H), an aryl group, a heteroaryl group, a cycloalkyl group or a heterocycloalkyl group, wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and —C(═O)OR⁵¹; wherein the heteroaryl group, is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl and —C(═O)OR⁵¹; where the cycloalkyl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and —C(═O)OR⁵¹; and wherein the heterocycloalkyl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl and —C(═O)OR⁵¹; R³⁰ is a C₁-C₆alkyl, or an aryl group, wherein the C₁-C₆alkyl group is optionally substituted with —NH2, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, one, two or three fluorine or chlorine atoms, or a hydroxyl group; and wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), and —N(C₁-C₆)dialkyl; R³¹ is a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); or a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R³² is a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); or a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); R²⁰ and R²¹ each independently represents a hydrogen atom; a C₁-C₆alkyl group which may be substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); a C₃-C₆cycloalkyl group which may be substituted with one or more fluorine or chlorine atom(s) or hydroxyl group(s); —C₁-C₆alkyldiyl(CO₂H) or together form a C₃-C₁₀-heterocycloalkyl which may be substituted with one or more C₁-C₆alkyl group(s), C₁-C₆alkoxy group(s), fluorine or chlorine atom(s) or hydroxyl group(s); R²² is a hydrogen atom, a C₁-C₆alkyl group; or a C₃-C₆cycloalkyl group; wherein the C₁-C₆alkyl group or the C₃-C₆cycloalkyl group is optionally substituted with —NH2, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxyl, or C₁-C₆alkoxy, an aralkyl group, a heteroalkyl group or a heteroalkylcycloalkyl group; R²³ is —OH, —O(C₁-C₃)alkyl, or —N(C₁-C₃)dialkyl; i is an integer of from 1 to 10; R²⁴, R²⁵, and R²⁶ each independently represents a hydrogen atom; —C(═O)C₁₁-C₂₁alkyl; or —C(═O)C₁₁-C₂₁alkenyl; R²⁷ represents —OH; —O(CH₂)₂NH₂, —OCH₂—[CH(NH₂)(CO₂H)], —O(CH₂)₂N(CH₃)₃; or

Xaa represents Gly, a conventional D,L-, D- or L-amino acid, a non-conventional D,L-, D- or L-amino acid, or a 2- to 10-mer peptide; and is joined to —C(═O) by an amide bond; o is an integer of from 1 to 10; R⁴ is selected from the group consisting of:

h is 0, 1, or 2; R⁵ represents a hydrogen atom; a fluorine or chlorine atom; —CF₃; —C(═O)OR⁵¹; —NHC(═O)R⁵²; —C(═O)NR⁵³R⁵⁴; or —S(O₂)OH; R⁵¹ represents a hydrogen atom; a C₁-C₆alkyl group; or a C₃-C₆cycloalkyl group; wherein the C₁-C₆alkyl group or the C₃-C₆cycloalkyl group is optionally substituted with —NH₂, NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxyl, or C₁-C₆alkoxy; R⁵², R⁵³ and R⁵⁴ each independently represents a C₁-C₆alkyl group which is optionally substituted with one or more fluorine or chlorine atom(s); a C₃-C₆cycloalkyl group which is optionally substituted with one or more fluorine or chlorine atom(s); or an aryl group which is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent; R⁶ and R⁷ each independently represents a hydroxyl group; an —O(C₁-C₆)alkyl group, an —O(C₂-C₆)alkenyl group, a, —O(C₁-C₆)alkyldiylO(C═O)(C₁-C₆)alkyl group, or a —O(C₁-C₆)alkyldiylO(C═O)(C₂-C₆)alkenyl group; wherein the C₁-C₆alkyl group and the C₂-C₆alkenyl group may be substituted with NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆ alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, or one, two or three fluorine or chlorine atom(s); or R⁶ represents a hydroxyl group and R⁷ represents a group:

R⁹ represents C₁-C₆alkyl, or aryl; wherein the C₁-C₆alkyl is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxy, C₁-C₆alkoxy, aryl, aryloxy, —C(═O)-aryl, —C(═O)C₁-C₆alkoxy; and wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent; g is 1 or 2; X¹ represents an oxygen atom; sulfur atom; or NH; X² represents an oxygen atom; sulfur atom; NH; or N(CH₃); X³ represents an oxygen atom; sulfur atom; nitrogen atom; carbon atom; or C—OH; and the dashed line represents a carbon-carbon bond or a carbon-carbon double bond; E is a group represented by the general formula (III) or (IV):

wherein R¹² and R¹³ are in cis configuration, and wherein ring A in formula (III) represents a 5-membered or 6-membered carbocyclic or heterocyclic ring containing at least one double bond, including an aromatic carbocyclic or heterocyclic ring, which can be substituted with one to three or one to four substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), and —N(C₁-C₆)dialkyl; and L and T each independently represents a ring atom, wherein L and T are adjacent to another; R¹² and R¹³ each independently represents a hydrogen atom, a fluorine atom, hydroxyl, —NH₂, C₁-C₆alkyl, C₁-C₆alkoxy, —C(═O)-aryl, —C(═O)C₁-C₆alkyl, or —SO₂(C₁-C₆alkyl); or —SO₂aryl; wherein any of the foregoing C₁-C₆alkyl, C₁-C₆alkoxy, or aryl are optionally substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; or R¹² and R¹³ are taken together to form a 5-membered or 6-membered ring, which ring is optionally substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; I is —(CH₂)_(m)—Y, wherein m is an integer of from 3 to 6, provided that m is an integer of from 3 to 5 when E is a group according to general formula (III); Y represents a group selected from the group consisting of:

wherein R⁴⁰, R⁴¹, R⁴³, R⁴⁴, R⁴⁶, R⁴⁸ and R⁴⁹ each independently represents a hydrogen atom, —C₁-C₆alkyl, —C₃-C₆cycloalkyl, —C₁-C₆alkoxy, —C(═O)aryl, or —C(═O)C₁-C₆alkyl, wherein any of the foregoing C₁-C₆alkyl, C₃-C₆cycloalkyl, C₁-C₆alkoxy, or aryl are optionally substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxy; or R⁴⁰ and R⁴¹, or R⁴³ and R⁴⁴, are taken together to form a 5-membered or 6-membered ring, which ring may be substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; R⁴², R⁴⁵, R⁴⁷ and R⁵⁰ each independently represents a —C₁-C₃alkyl, wherein the C₁-C₃alkyl may be substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₃)alkyl, —N(C₁-C₃)dialkyl, C₁-C₃alkylcarbonyloxy-, C₁-C₃alkoxycarbonyloxy-, C₁-C₃alkylcarbonylthio-, C₁-C₃alkylaminocarbonyl-, di(C₁-C₃)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; or R⁴⁰ and R⁴¹; R⁴³ and R⁴⁴; R⁴⁹ and R⁵⁰ are taken together to form a 5-membered or 6-membered ring, which ring may be substituted with one, two or three substituents independently selected from the group consisting of —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, C₁-C₆alkylcarbonyloxy-, C₁-C₆alkoxycarbonyloxy-, C₁-C₆alkylcarbonylthio-, C₁-C₆alkylaminocarbonyl-, di(C₁-C₆)alkylaminocarbonyl-, fluorine or chlorine atom, and hydroxyl; f is an integer of from 0 to 2; with the proviso that when X does not comprise a —C(═O)O-motif with the carbonyl carbon in alpha or beta position to the oxygen atom of general formula (II), Y is an oxamide, a carbamate or a carbamide as defined above.
 2. The compound according to claim 1, with the further proviso that when n is 3, 5, 6, 7 or 8, k is 1 and E is a group according to general formula (III) or general formula (IV), wherein each of R¹² and R¹³ is a hydrogen atom; P represents a group: —(CH₂)₃—O—(CH₂)—X⁸¹; —(CH₂)₅—O—(CH₂)—X⁸¹; wherein X⁸¹ represents a group selected from the group consisting of:

R^(1′) is defined as R¹ above; R^(2′) represents —NHR^(3′); —OR^(22′); —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O—, 3-O—, or 6-O-position of the saccharide; or wherein R² is selected from the group consisting of:

wherein R^(3′) represents (SO₂R³⁰); (OR³¹); —C₁-C₆alkanediyl(SO₂R³²); or —C₂-C₆alkanediyl(CO₂H); R^(22′) is a hydrogen or a C₃-C₆cycloalkyl group, which is optionally substituted with —NH₂, —NH(C₁-C₆)alkyl, —N(C₁-C₆)dialkyl, —NH(C₁-C₆)alkyldiyl-C₁-C₆alkoxy, one, two or three fluorine or chlorine atom(s), hydroxy, or C₁-C₆alkoxy; R²³ and i are as defined above; R²⁴, R²⁵, R²⁶, and R²⁷ are as defined above; R^(4′) is defined as R⁴ above; and h is defined as above; R^(6′) and R^(7′) are defined as R⁶ and R⁷ above; R^(9′) is defined as R⁹ above; R^(9″) represents aryl which is optionally substituted with one, two or three substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent.
 3. The compound according to claim 1, wherein X is

wherein R² is —OR²²; —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O-, 3-O-, or 6-O-position of the saccharide; or wherein R² is selected from the group consisting of:

wherein R²³ and i are as defined above; and wherein R²², and R²³ to R²⁷ are as defined in claim
 1. 4. The compound according to claim 1, wherein X is —C(═O)OH or a salt of the carboxylic acid.
 5. The compound according to claim 1, wherein Y is one of the oxamides defined according to claim
 1. 6. The compound according to claim 1, wherein X is

wherein R² is —OR²²; —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O-, 3-O-, or 6-O-position of the saccharide; or wherein R² is selected from the group consisting of:

wherein R²², R²³ to R²⁷ and i are as defined in claim 1, and wherein Y is one of the oxamides defined according to claim
 1. 7. The compound according to claim 1, wherein X is C(═O)OH, and Y is one of the oxamides defined according to claim
 1. 8. The compound according to claim 1, with the formula (V):

wherein R⁵⁵ represents —OH; —OR²²; —(OCH₂—CH₂)_(i)—R²³; a mono-, or disaccharide, or a derivative thereof, which is joined to —C(═O) by an ester bond via the 1-O-, 3-O-, or 6-O-position of the saccharide; R²², R²³ and i are as defined in claim 1; Y represents a group selected from the group consisting of:

wherein R⁴⁰ to R⁵⁰ are defined in claim 1; R⁵⁷ and R⁵⁸ are hydrogen; or form together a five- or six-membered ring, preferably an aromatic ring, optionally substituted with one to three or one to four substituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, fluorine or chlorine atom, hydroxyl group, amino group, —NH(C₁-C₆alkyl), —N(C₁-C₆)dialkyl, and an oxo substituent; s is 0, 1 or 2, with the proviso that if s is 0, R⁵⁷ and R⁵⁸ form together a five- or six-membered ring; the double bond in formula (V) represents a double carbon-carbon bond in cis-configuration, if R⁵⁷ and R⁵⁸ are hydrogen, or this double bond is part of a five- or six-membered ring formed together by R⁵⁷ and R⁵⁸.
 9. The compound according to claim 8, wherein R⁵⁵ represents —OH or —(OCH₂—CH₂)_(i)—R²³; i is 2 to 4; Y is an oxamide, a carbamide or a carbamate; R⁵⁷ and R⁵⁸ are both H, or together form a substituted or non-substituted five- or six-membered aromatic ring; and s is 1 or s is 0 if R⁵⁷ and R⁵⁸ together form a substituted or non-substituted five- or six-membered aromatic ring.
 10. The compounds according to claim 1, wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 11. The compound according to claim 1, with the formula (VI)

or a pharmaceutically acceptable salt thereof.
 12. A pharmaceutical composition comprising the compound according to claim 1 in combination with a physiologically acceptable excipient.
 13. A method for the treatment of a cardiovascular disease, comprising the step of administering a compound according to claim 1 to a subject in need thereof in an effective amount.
 14. The method according to claim 13, wherein the cardiovascular disease is selected from the group consisting of atrial fibrillation, atrial tachycardia, ventricular arrhythmia, and heart failure.
 15. The method according to claim 13, wherein the compound or composition is administered orally, topically, subcutaneously, intramuscularly, intraperitoneally, intravenously, intranasally.
 16. The method according to claim 13, wherein the compound or composition is a dosage form selected from the group consisting of a spray, an aerosol, a foam, an inhalant, a powder, a tablet, a capsule, a soft gelatin capsule, a tea, a syrup, a granule, a chewable tablet, a salve, a cream, a gel, a suppository, a lozenge, a liposome composition and a solution for injection.
 17. The method according to claim 13, wherein the cardiovascular disease is selected from the group consisting of atrial fibrillation, ventricular arrhythmia, heart failure, coronary artery disease, myocardial infarction, maladaptive cardiac hypertrophy, and cardiac arrhythmias including ventricular extrasystoles, ventricular tachycardia, malignant ventricular tachycardia, atrial tachycardia, atrial flutter and atrial fibrillation, dilatative cardiomyopathy, and hypertensive heart disease.
 18. A method for the treatment of a cardiovascular disease, comprising the step of administering a composition according to claim 12, to a subject in need thereof in an effective amount.
 19. The method according to claim 18, wherein the cardiovascular disease is selected from the group consisting of atrial fibrillation, ventricular arrhythmia, heart failure, coronary artery disease, myocardial infarction, maladaptive cardiac hypertrophy, and cardiac arrhythmias including ventricular extrasystoles, ventricular tachycardia, malignant ventricular tachycardia, atrial tachycardia, atrial flutter and atrial fibrillation, dilatative cardiomyopathy, and hypertensive heart disease. 